Pharmaceutical applications of chitosan

Accepted Manuscript Pharmaceutical applications of chitosan

Zahra Shariatinia PII: DOI: Reference:

S0001-8686(18)30277-X https://doi.org/10.1016/j.cis.2018.11.008 CIS 1927

To appear in:

Advances in Colloid and Interface Science

Please cite this article as: Zahra Shariatinia , Pharmaceutical applications of chitosan. Cis (2018), https://doi.org/10.1016/j.cis.2018.11.008

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ACCEPTED MANUSCRIPT Pharmaceutical applications of chitosan

Zahra Shariatinia [email protected]

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Department of Chemistry, Amirkabir University of Technology (Tehran Polytechnic),

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Corresponding author.

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

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P.O.Box:15875-4413, Tehran, Iran

ACCEPTED MANUSCRIPT Abstract Chitosan (CS) is a linear polysaccharide which is achieved by deacetylation of chitin, which is the second most plentiful compound in nature, after cellulose. It is a linear copolymer

of β-(1→4)-linked

deoxy-β-D-glucopyranose.

It

2-acetamido-2-deoxy-β-D-glucopyranose and has

appreciated

properties

such

as

2-amino-2-

biocompatibility,

biodegradability, hydrophilicity, nontoxicity, high bioavailability, simplicity of modification, favorable permselectivity of water, outstanding chemical resistance, capability to form films,

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gels, nanoparticles, microparticles and beads as well as affinity to metals, proteins and dyes. Also, the biodegradable CS is broken down in the human body to safe compounds (amino

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sugars) which are easily absorbed. At present, CS and its derivatives are broadly investigated

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in numerous pharmaceutical and medical applications including drug/gene delivery, wound dressings, implants, contact lenses, tissue engineering and cell encapsulation. Besides, CS has

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several OH and NH2 functional groups which allow protein binding. CS with a deacetylation degree of ~50% is soluble in aqueous acidic environment. While CS is dissolved in acidic medium, its amino groups in the polymeric chains are protonated and it becomes cationic

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which allows its strong interaction with different kinds of molecules. It is believed that this positive charge is responsible for the antimicrobial activity of CS through the interaction with

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the negatively charged cell membranes of microorganisms. This review presents properties and numerous applications of chitosan-based compounds in drug delivery, gene delivery, cell encapsulation, protein binding, tissue engineering, preparation of implants and contact lenses, healing,

bioimaging,

antimicrobial food

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wound

additives,

antibacterial food

packaging

materials and antibacterial textiles. Moreover, some recent molecular dynamics simulations

Keywords:

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accomplished on the pharmaceutical applications of chitosan were presented.

Chitosan;

Pharmaceutical

applications;

engineering; Wound healing; Antimicrobial activity

Drug

delivery

systems;

Tissue

ACCEPTED MANUSCRIPT 1. Introduction Chitosan (CS) is the most important derivative of chitin which is prepared by the alkaline deacetylation of chitin [1]. Chitosan structure is comprised of β-1,4-linked 2-amino2-deoxy-β-D-glucose (deacetylated D-glucosamine) and N-acetyl-D-glucosamine units [2]. According to United States Food and Drug Administration (USFDA), it is a GRAS (Generally Recognized as Safe) material and therefore it has found wide pharmaceutical and biomedical applications [3]. It is known as a bioactive compound that has shown numerous biological

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properties such as antitumor, immunoenhancing, antifungal, antimicrobial, antioxidant and wound healing activities. These features plus some exceptional properties such as non-

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toxicity, biodegradability, biocompatibility, non-antigenic and low-cost have led to its

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extensive pharmaceutical applications including biomedicine with the possibility of clinical use [4], drug delivery systems [5-8], tissue engineering [9], food technology [10,11],

wound healing [17] and textile industry [18].

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bioimaging [12], implants [13], contact lenses [14], gene delivery [15], protein binding [16],

In this review, the key research findings on the most recent pharmaceutical applications

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of CS in diverse biomedical fields such as wound healing, tissue engineering, drug/gene delivery, protein binding, cell encapsulation, preparation of implants and contact lenses,

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bioimaging, food additives, food packaging and antibacterial textiles (Scheme 1) will be presented. The molecular dynamics simulations on the pharmaceutical applications of

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chitosan were also afforded.

2. Pharmaceutical application of chitosan in medicine 2.1. Application of chitosan in pharmaceutics/drug delivery pharmaceutical

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Recently,

carriers

such

as

polymers,

micelles,

liposomes

and

nanoparticles have received increased attention [19-22]. These systems reveal numerous advantages principally in enhanced efficacy and safety of the drugs. These systems can incorporate both hydrophobic and hydrophilic active compounds, which depends on the carrier nature. As well, they can provide higher stability for the therapeutics against chemical and enzymatic degradation, longer drug influence in the target tissue, superior bioavailability and drug targeting by inclusion of specific ligands [23]. These systems are used to carry small active molecules,

proteins,

peptides,

vaccines, genes and oligonucleotides which are

adsorbed, encapsulated, covalently and/or electrostatically attached to their surface or within their matrix [24]. Drug delivery systems based on polysaccharides have revolutionized medical treatments due to their efficient and appropriate drug delivery. Among various

ACCEPTED MANUSCRIPT polymers, the CS-based drug delivery systems are attracting substantial interest as the vehicles that are able to release their contents at the desired rate and location in the body [25]. In a recent study, aceclofenac-loaded nanocomposites were prepared using two natural polysaccharides including CS and locust bean gum (LBG) and glutaraldehyde as cross-linker [28]. The infrared spectra established the formation of composites and the chemical compatibility of polymers and drug. The influence of polymeric components on the particle size and drug entrapment efficiency (DEE) of the composites was investigated. It was shown

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that increasing LBG amount really lessened the DEE from 72% to 40% and created larger particles (372-485 nm). Nonetheless, a reverse trend was observed when the CS concentration

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was enhanced. Among these composites, the greatest DEE=78.92% and the smallest

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composites size (318 nm) was gained when LBG:CS mass ratio was 1:5. Also, the CS:LBG (1:5) exhibited the slowest drug release rate in phosphate buffer solution (pH=6.8) up to 8 h.

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The drug release properties excellently supported the swelling results of the nanocomposites so that the composites proficiently suppressed the burst release of drug in acidic solution (pH=1.2). The in vitro drug delivery by the nanocomposites was happened through anomalous

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transport mechanism. Hence, these CS and LBG nanocomposite systems could decrease the gastrointestinal side effects of the drug through slow sustained release of the medication.

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CS nanoparticles (NPs) were utilized for the encapsulation of levofloxacin antibacterial agent to treat ocular infection [29]. The CS NPs were obtained through ionic gelation process using CS and sodium tripolyphosphate. The formulations displayed the particle size in

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nanometer range with high loading and encapsulation efficiency. The optimized formulation was changed to a sol-gel system in order to increase the corneal residence time. The histopathology of cornea established that the optimized formulation was non-irritant and

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benign for topical ophthalmic usage. Hen's egg test-chorioallantoic membrane (HET-CAM) test is a rapid, inexpensive and sensitive method used to check the irritation ability of ophthalmic formulations. The chorioallantoic membrane of the chick embryo contains complete tissue which includes arteries, veins and capillaries that technically responds to injuries with a complete inflammatory process. The LFX-CS-NPs, sodium chloride solution (0.9%, negative control) and 0.1 N NaOH (positive control) were examined for their irritation potentials so that the irritation scores were obtained in 5 min (Fig. 1). It was shown that both of the LFX-CS-NPs and sodium chloride solution were almost non-irritating (score 1) but 0.1N NaOH solution exhibited coagulation which confirmed this solution caused irritating effects (score 13.43). Thus, the non-irritant LFX-CS-NPs in situ gel system could be well tolerated for ocular administration [29].

ACCEPTED MANUSCRIPT The antimicrobial assay proved that the formulation had high antibacterial effect against S. aureus and P. aeruginosa microorganisms. The pharmacoscintigraphic test showed the decreased corneal clearance, nasolachrymal drainage and greater LFX retention compared to LFX solution. It was determined that the levofloxacin loaded CS NPs in situ gel system was a proficient vehicle for ocular levofloxacin delivery. Histopathology of goat eye cornea did not illustrate any variations in the eye tissue after the optimized LFX-CS-NPs in situ gel formulation was applied that verifies the LFX-CS-NPs was safe for ocular application without

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any ocular irritation influence. It did not display any histopathological changes so that the cells in the corneal membrane were remained intact in the same position without any rupture

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in the corneal section in comparison to the control (Figs. 2A and 2B) [29].

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Confocal laser scanning microscopy (CLSM) of the formulations on excised goat cornea showed the permeation extent using the fluorescent intensity. The dye loaded LFX-

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CS-NPs in situ gel penetration depth into the cornea was 91.73 µm having a high fluorescence intensity compared to that of the LFX solution (47.65 µm depth), see Figs. 3A and 3B. High penetration of LFX-CS-NPs can be associated to the bioadhesive nature and high viscosity of

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CS which caused extended residence time on the cornea, hence intracellular and intercellular penetration was occurred [29].

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In another research, negatively charged carboxylic curdlan (Cc) including a β-1,3polyglucuronic acid structure was used to produce nanosized polyelectrolyte complexes (PECs) with positively charged CS in aqueous solution as effective delivery carriers for

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anticancer drug 5-fluorouracil (5Fu) [30]. Nanosized CS/Cc PECs were prepared by adding 0.5 mg/mL solutions of CS and Cc in 1:1 (w/w) ratio at pH=3.0. Under the optimized conditions, the CS-Cc PECs revealed an average size of around 180 nm, spherical

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morphology and a zeta potential of about 41 mV. The 5Fu drug was loaded to the nanosized CS/Cc PECs and exhibited outstanding loading content (10.81%) and encapsulation efficacy (86.47%). The in vitro drug release data specified that the nanosized CS-Cc PECs were potential platforms for the sustained release of 5Fu having an anomalous transport mechanism which followed the Ritger–Peppas model. Moreover, the CS-Cc PECs demonstrated low in vitro cytotoxic activity against HeLa and SPCA-1 cell lines. Thus, it was suggested that the developed nanosized CS-Cc polyelectrolyte complexes were promising antitumor drug vehicles. The in vitro drug release was examined using two pH values (1.4 and 7.4) at 37 C for 2 days. Fig. 4 displays the cumulative 5Fu release profiles from the CS/Cc PECs. It was found that the 5Fu was released from the CS/Cc PECs in a sustained pH-dependent manner. The

ACCEPTED MANUSCRIPT controlled 5Fu release from the PECs was related to the interaction of 5Fu and the –COOH functionalities existing on the PECs. Also, the burst 5Fu release was happened in the first 12 h which was followed by a steadily and slightly controlled release. The cumulative 5Fu release after 48 h was nearly 87% for pH 7.4 and 70% for pH 1.4. The cumulative 5Fu release was greater in neutral pH medium than in acidic pH. The zeta potential values of both the blank and 5Fu-loaded CS/Cc PECs were positive confirming CS dominated the PECs and placed on the PECs surface. In acidic condition, the CS chains at the interface are completely expanded

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that create an interface to decrease the drug diffusion out of the nanocarrier. In neutral pH medium, the deprotonated CS chains at the interface are collapsed to the colloids cores and

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could not affect the drug diffusion. Furthermore, the greater interactions of 5Fu and the CS/Cc

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PECs functional groups (particularly –COOH) are probably responsible for the enhanced 5Fu release from the PECs in neutral pH [30].

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Nowadays, nanoparticle-based vaginal drug delivery carriers have received great attraction for the administration of peptide based-vaccines or microbicides in order to inhibit and/or treat sexually transmitted diseases [31]. In this context, a direct and effective approach

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was established for the vaginal application and delivery of peptide-loaded mucoadhesive nanoparticles [31]. This formulation was principally consisted of CS NPs encapsulated in

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appropriate hydrophilic freeze-dried cylinders. The CS nanoparticles were used to carry the peptide drug and allow adhesion to the vaginal mucosal epithelium. Hydrophilic freeze-dried cylinders assisted the fats release of the nanoparticles to the vaginal zone. After contacting

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with the aqueous vaginal medium, the excipients of these sponge-like materials were rapidly dissolved and facilitated the release of their contents. The in vitro release test displayed the capability of the sponge-like systems and CS NPs to deliver the mucoadhesive nanoparticles

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and peptide, respectively. The confocal laser scanning micrographs verified that the nanoparticles were able to promote the peptide penetration into the vaginal mucosa. Thermoresponsive nano-sized hydrogel particles were prepared as smart platforms using CS natural polymer for curcumin delivery [32]. Chitosan backbone was grafted to poly– (N-isopropylacrylamide)

(pNIPAM)

through

(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide

the

well-known coupling

reaction.

N-ethyl-N′-(3The

CS-

grafted pNIPAM (CS-g-pN) nanogels were obtained by a sonication process. Addition of curcumin to the CS-g-pN nanogels was done by incubation route. Size, morphology of nanogels, curcumin amounts in the nanogels and cellular uptake were examined by DLS, TEM, fluorescent spectroscopy and confocal microscopy techniques, respectively. The CellTiter-Blue® cell viability assay was carried out using HeLa and NIH-3T3 cells to

ACCEPTED MANUSCRIPT evaluate the safety and the MTT assay was performed using HepG2, Caco-2, HT-29 and MDA-231 cells to determine cytotoxic properties. It was shown that CS-g-pN with 3–60% modification degree was easily assembled as curcumin encapsulated spherical nanogel particles with submicron sizes. The thermoresponsive performances of CSg-pN nanogel formulations were different because grafted pNIPAM had various length and density. The CS-g-pN nanogels were non-toxic to HeLa and NIH-3T3 cells. All of curcumin-loaded CS-gpN nanogel were uptaken to NIH-3T3 cells and exhibited dose-dependent cytotoxicity against

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cell lines tested. Therefore, development of such curcumin-loaded nanogels produced materials that could be functionalized and applied for targeted therapy and controlled delivery

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of small drug molecules and biomolecules for biomedical applications.

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Folic acid-CS conjugates were used as drug delivery tools by loading of folic acid (FA) by chitosan-15 and chitosan-100 kDa in aqueous solution at physiological pH [33]. Thermodynamic parameters ΔH =−18 to −12 (kJ·mol−1 ), ΔS = −22 to −8 J.mol−1 .K−1 and ΔG

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= −11 to −9 kJ.mol−1 displayed that FA-CS bindings occurred through van der Waals and Hbonding contacts. The FA-CS conjugate stability was enhanced by increasing the polymer

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size. The FA loading efficiency was 35 to 55%. The TEM images demonstrated great polymer morphological variations upon acid interaction. The CS nanoparticles were able to in vitro

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deliver folic acid.

It is well-known that in cancer theranostics, the key strategy in nanoparticle-based targeted

delivery system is enhanced permeability and retention (EPR) influence of

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macromolecules [34]. Application of varied nanoparticles offers a deeper understanding of diverse EPR effects which depends on their structures, chemical modifications and physicochemical features. Currently, the tumor microenvironment is considered as another

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significant factor in tumor-targeted delivery by nanoparticles but the relationship between EPR effects and tumor microenvironment has not yet been completely clarified. Thus, ectopic subcutaneous tumor models representing dissimilar tumor microenvironments were prepared by inoculation of U87, SCC7, HT29, A549 and PC3 cancer cell lines to athymic nude mice [34]. The tumor-targeted delivery of self-assembled glycol CS nanoparticles was tested using the five diverse types of tumor-bearing mice in order to recognize the correlation of the tumor microenvironments with targeted delivery of CS NPs. The neovascularization and degree of intratumoral extracellular matrix (ECM) were both vital in defining the tumor targeted delivery of CS NPs. The EPR influence was increased in the tumors including great amount of angiogenic blood vessels and little intratumoral ECM content. Thus, it was found that different EPR impact was achieved for the tumor-targeted delivery of nanoparticles and it

ACCEPTED MANUSCRIPT depended on the tumor microenvironment in distinct tumors. In order to overcome existing limits in clinical nanomedicine, the tumor microenvironment of the patients and EPR effects in clinical tumors must also be examined [34]. The in vitro CNPs cellular uptake was comparatively assessed in several cancer cells in three pH environments (pH 6.0–7.4). In pH 6.0, 6.5 and 7.4, the CNPs were observed at the cancer cells membranes after 30 min of incubation (Fig. 5). Besides, the in vitro CNPs cellular uptake was time dependently tested using five different cancer cell lines. After 6 and

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24 h post-incubation, the near-infrared fluorescence (NIRF) signal representing the CNPs distribution were detected in the cytoplasms of the cancer cells. Thus, the CNPs were

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effectively internalized to the cancer cells confirming the CNPs were delivered to these five

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diverse cancer cells in vitro [34].

The targeting efficiency of CNPs in dissimilar tumor tissues was assessed. The Cy5.5-

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labeled CNPs (1 mg/mL, 200 μL/head) were injected intravenously to the tumor-bearing mice with the mean volume of each tumor was ~300–350 mm3 . The time-dependent whole body NIRF images indicated the in vivo CNPs distribution in the mice that were transplanted with

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five different cancer cells, Fig. 6. The NIRF intensities at tumor sites were steadily enhanced and the maximum intensities from tumors were achieved between 24 and 72 h. also, the

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whole body NIRF images established that the CNPs were circulated in the blood during a long time and be successfully delivered to tumors in a targeted way. Even though the CNPs were accumulated in the tumor cells of all tumor-grafted mice, the targeting effectiveness of

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CNPs was significantly dissimilar which was depended on the tumor type. The CNPs provided high contrast images for SCC7, U87 and HT29 tumors confirming they were efficiently delivered to the tumors. However, PC3 and A549 tumors provided rather low

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NIRF signals demonstrating few CNPs were delivered to these tumors [34]. The tumors encapsulated with fibrous connective tissue was totally stained blue using trichrome stain and the peritumoral area was not used for the imaging in order to eliminate such area of excess collagen in dermis. The ECM contents in the tumor tissues were observed and each tumor type displayed diverse amounts of intratumoral ECM, Fig. 7A. The A549 and PC3 tumors exhibited particularly high collagen amounts in the tumor nests (>2.0%) but the SCC7 and HT29 tumors contained rather smaller ECM amounts (<0.51%). The SCC7, U87 and HT29 tumors revealed considerably smaller ECM amounts compared to the PC3 and A549 tumors (see Fig. 7B). As the ECM amount in tumor tissues was increased, the tumortargeted delivery efficacy of CNPs was decreased. It was suggested that the intratumoral ECM might be a physical barrier to inhibit the tumor-targeted CNPs delivery [34].

ACCEPTED MANUSCRIPT A mucoadhesive polymer was synthesized by conjugation of CS to poly(ethylene glycol) diacrylate (PEGDA) through the Michael type reaction [35]. A higher PEGDA grafting efficacy was observed using low molecular weight PEGDA (0.7 kDa) compared to long 10 kDa PEGDA. The acrylation percentage was determined based on the ninhydrin reaction with CS which approved the data measured from the NMR spectra. The adhesive properties were assessed by tensile analysis and rotating system which involved detachment of polymer tablets from a fresh intestine sample. The CS modified with high molecular

modified

and

thiolated

CS.

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weight PEGDA revealed improved mucoadhesive characteristics in comparison to both nonThe rheology measurements of polymer/mucin mixtures

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confirmed that strong interactions occurred between modified CS and mucin glycoproteins.

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Hence, this formulation could be used as a beneficial polymeric delivery system to afford prolonged residence time for the vehicle on the mucosa surface.

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The cysteine conjugated CS/PMLA multifunctional nanoparticles were synthesized to be used as targeted drug delivery systems in order to eliminate Helicobacter pylori [36]. Helicobacter pylori specifically expresses urea transport protein on its membrane in order to

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transfer urea to the cytoplasm urease for supplying ammonia that protects bacteria against the acidic environment of the stomach. Appropriate clinical topical antimicrobial agents are

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necessary to discard Helicobacter pylori from the inflamed basal area. For this purpose, cysteine conjugated CS (Cys-CS) derivatives were obtained for their mucoadhesive and anticoagulant features to prepare multifunctional nanoparticles. The technique was optimized

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to acquire Cys-CS/PMLA nanoparticles for amoxicillin encapsulation. Results displayed that amoxicillin-Cys-CS/PMLA nanoparticles were satisfactorily pH-sensitive that could delay the amoxicillin release at gastric acid and deliver the drug in order to efficiently target the

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Helicobacter pylori at its survival region. Compared to unmodified amoxicillin-CS/PMLA nanoparticles,

efficient

inhibition

amoxicillin-Cys-CS/PMLA

of Helicobacter

nanoparticles.

Thus,

pylori the

growth was witnessed

multifunctional

for

amoxicillin-loaded

nanoparticles had shown high potency to effectively treat the Helicobacter pylori infection. Also, these pharmacologically powerful nanocarriers could be appropriate candidates for oral targeted delivery of diverse therapeutics/drugs for the treatment of Helicobacter pylori. The surface charge necessitating for nanoparticles to have extended circulation and good cell affinity have led to develop various polymeric nanoparticles for controlled drug delivery [37]. Recently, vancomycin loaded composite nanoparticles with a CS core and poly(lactide-co-glycolide) (PLGA) shell structure were fabricated that were pH-responsive and had surface charge switchable properties [37]. The spherical nanocomposites were

ACCEPTED MANUSCRIPT obtained by a modified emulsion-gelation technique with a controlled surface charge (-27.631.75 mV), size (316-573 nm) and encapsulation efficiency (up to 70.8%). The composite nanoparticles had particularly designed core-shell structures that were negatively charged in the beginning and switched to positively charge afterward. The negative charge of particles was slowly switched to positive charge due to the erosion of biodegradable polymer shells and contact of the positively charged CS core. The CS hydrogels displayed multi-layer structures which were mostly affected by CS concentration. Effects of the CS gelation

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behavior on the characteristics of the nanocomposites in reaction to various CS and NH3 concentrations were examined. Release rate was significantly declined by increasing CS

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concentration. After the CS incorporation, the enhanced drug release rate was seen by orders

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of magnitude for the samples immersed in the phosphate buffer saline solution with lower pH value which proved a pH responsive release feature. The drug release plots of the

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nanocomposites were composed of fast and slow release stages. The fast release was fitted with a modified first-order kinetic model but the slow release stage was described by the classical first-order release kinetic model.

Such results justified that the composite

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nanoparticles were promising systems for application in controlled drug delivery. The key issues in drug delivery to the oromucosa include boosting the mucoadhesion of

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drug formulations in order to elongate their residence time on the target site and the sustained drug release from the formulations [38]. In a recent investigation, the mucoadhesive CS biopolymer and its derivatives, including high and low molecular weights as well as a

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carboxylic derivative, were used as the excipients to obtain spray-dried microspheres for the oromucosal benzydamine hydrochloride drug delivery to enhance the delivery effectiveness [38]. All CS modified spray-dried powders displayed greater surface roughness, sizes below

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10 μm, moisture content below 10% w/w and better flowability. Addition of CS to the formulations extremely extended the drug release duration. Besides, the mucoadhesive interaction of CS-modified microparticles was highly increased and it was further improved by enhancement of CS the content in the formulations. Thus, it was demonstrated that adding CS to the spray-dried microspheres elongated the drug release duration and enhanced the mucoadhesive interaction force of microspheres to the mucosal membrane, which could be advantageous in the treatment of patients. In another research, the probability of preparing multifunctional oral insulin delivery systems was examined through conjugation of different dipeptides on CS and trimethyl CS to be employed as drug carriers [39]. The conjugates of glycyl-glycine and alanyl-alanine of CS and trimethyl CS (on primary alcohol group of polymer located on carbon 6) were

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and nanoparticles including insulin were achieved for oral delivery. The

nanoparticles preparation conditions were optimized and their performances were assessed to improve the insulin permeability and cytotoxicity of nanoparticles against Caco-2 cell line. In order to estimate the effectiveness of orally administered nanoparticles, the nanoparticles with the most permeability were investigated in male Wistar rats as animal model and their insulin and glucose serum levels were measured. For all the conjugates, the IR and NMR spectra established their successful formation with the favorite substitution degree. Under optimizing

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preparation conditions, nanoparticles with anticipated size (157.3–197.7 nm), polydispersity index (0.365–0.512), zeta potential (24.35–34.37 mV), loading capacity (30.92–56.81%),

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entrapment efficiency (70.60–86.52%), suitable morphology and appropriate release pattern

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were attained. Glycyl-glycine and alanylalanine conjugate nanoparticles of trimethyl CS presented 2.5–3.3 folds more efficient insulin permeability in Caco-2 cells than their CS In

animal model,

oral administration of glycyl-glycine and

alanyl-alanine

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analogues.

conjugate nanoparticles of trimethyl CS established rational increase in serum insulin level with relative bioavailability of 17.19 and 15.46% for glycyl-glycine and alanyl-alanine

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conjugate nanoparticles, respectively. Also, serum glucose level was decreased compared to trimethyl CS nanoparticles. The glycyl-glycine and alanyl-alanine conjugate nanoparticles of

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trimethyl CS could orally deliver insulin by more than one mechanism in the animal model. Therefore, they would be identified as auspicious candidates for biomedical applications. In order to increase the bioavailability, aqueous stability, developing suitable delivery

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systems, and enhancing the chemotherapeutic influence of curcumin, it was encapsulated in CS-based polyelectrolyte complexes and their antidiabetic activities were evaluated through in vitro α-amylase inhibition test and in vivo antihyperglycaemic potency in alloxan-induced

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rats [40]. The microscopic analysis revealed curcumin nanoformulations (<50 nm) that were loaded in CS-alginate complex. Higher encapsulation efficiency (64–76%), loading capacity (20–26%) and yield (50–72%) were attained. Complexation of CS with alginate decreased the curcumin loss (by 20%) and prolonged mean release time (by 40 min) in simulated gastric fluid. The time dependent experiment exhibited that the α-amylase inhibition property of curcumin was improved by the nanoencapsulation. It was indicated that oral administration of sub-therapeutic dosage of curcumin (50 mg/kg b.wt.) nanoencapsulated in CS-based complexes considerably decreased hyperglycaemia after 7 days of treatment. Finally, the chemotherapeutic efficacy of curcumin was improved by its nanoencapsulation in CS-based polyelectrolytes.

ACCEPTED MANUSCRIPT It is known that most currently existing chemotherapy methods for cancer treatment have limited to clinical cancer therapies which is primarily because of low drug encapsulating efficacy and little pharmacologically effective concentrations at the tumor sites [41]. Therefore, in a recent work, a simple approach was developed to increase drug encapsulating efficiency and local drug concentration by an injectable hydrogel prepared using thiolated chitosan (TCS) and poly(ethylene glycol) diacrylate (PEGDA) [41]. It was shown that nearly 100% encapsulating capacity was attained for the anti-cancer drug curcumin into this system.

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The curcumin interaction with PEGDA was recognized by fluorescence spectroscopy and the binding constant was calculated; then, this interaction was simulated with a docking

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computation. In order to enhance the antitumor activity and reach favored local concentration,

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lysozyme was added to the system. A sustained curcumin release was perceived with a controlled lysozyme-responsive performance that quickly led to the drug concentration

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reaching the therapeutic threshold. Such system exhibited proficient intracellular curcumin release in order to stimulate in vitro apoptosis of cancer cells. As well, the system successfully postponed the tumor growth and decreased adverse effects in tumor-bearing nude

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mice. Consequently, injectable hydrogel would be a beneficial system to be employed for encapsulation of various insoluble anticancer drugs.

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The intracellular curcumin release was evaluated using Hoechst 33,258 stain which is a nuclear counter stain emitting blue fluorescence once bound to DNA. It is employed for counterstaining, apoptosis and cell cycle tests. The living cells nuclei merely stain a

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homogeneous and weak blue color whereas the apoptotic cells nuclei stain bright blue so that the chromatin is condensed. It is observed in Fig. 8 that the nuclei of untreated cells were stained elliptical blue with the staining was diffused into the nuclei that displayed nearly no

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apoptotic nuclei existed in the untreated HepG2 cells (Fig. 8a). Treating with free curcumin (Fig. 8b) as well as the curcumin containing injectable hydrogels (Figs. 8c and 8d) lead to the apoptotic morphological variations including condensed chromatin, shrunken and condensed bright blue nuclei which suggested HepG2 cells treatment with curcumin induced apoptosis. In comparison with the curcumin incorporated injectable hydrogels without lysozyme (Fig. 8c), additional apoptotic cells were created after 24 h incubation by curcumin containing injectable hydrogels with lysozyme (Fig. 8d) [41]. The in vivo antitumor activity of curcumin loaded TP3 was assessed in male nude mice through their comparison with saline control, free curcumin as well as curcumin incorporated TP0. For this purpose, the tumor volumes and body weights of tumor-bearing mice were measured every three days during 21 days (Fig. 9). In Fig. 9a, serious side effects were

ACCEPTED MANUSCRIPT witnessed in saline treated mice which was observed by the substantial body-weight loss (Fig. 9b). A noticeable body-weight loss was measured for the free curcumin treated tumor-bearing nude mice (Fig. 9b). Nevertheless, a small decline in body weight was observed in mice treated with curcumin containing TCS/PEGDA hydrogels which indicated they led to less side effects for the tumor treatment compared to the free curcumin. It is seen in Fig. 9c that the curcumin incorporated TP3 treated group demonstrated comparable tumor growth inhibition in vivo relative to the free curcumin treated group so that the tumor volumes of

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curcumin containing TP3 treated mice were very smaller than those of mice treated curcumin loaded TP0 or with saline. Moreover, the tumor volumes of the mice treated by curcumin

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loaded TP0 were smaller than those treated by saline. Thus, curcumin was efficiently released

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from hydrogels to inhabit tumor growth. As the tumor volumes of the curcumin incorporated TP0 treated mice were not as small as those treated with curcumin containing TP3, the

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curcumin loaded hydrogel with lysozyme showed rather greater tumor inhibition in vivo compared to the curcumin loaded hydrogel without lysozyme and this was in agreement with the in vitro tests. The tumor inhibition rates using the free curcumin, curcumin loaded TP0

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and curcumin loaded TP3 were measured to be 48.4%, 34.0% and 57.5%, respectively. Additionally, the tumor inhibition values of curcumin loaded TCS/PEGDA hydrogels (TP3)

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were greater than 40% which was considered as an effective treatment [41]. Histological analysis by hematoxylin and eosin (H&E) staining was performed in order to further assess the antitumor effectiveness and the possible toxicity of curcumin containing

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TP3 (Fig. 10). Numerous tumor cells were seen in the saline treated tumor tissues demonstrating fast tumor growth. Nonetheless, pyknotic cells with highly condensed nuclei existing in the curcumin incorporated TP3 treated tumor tissues showed much more dead or

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apoptotic cells. Large necrotic areas found in the curcumin treated tumor tissues exhibited the tumor growth inhibition by the curcumin containing TP3. As well, tissue sections from curcumin loaded TP3 treated mice organs were evaluated for their probable toxicity. It is seen in Fig. 10 that there were no apparent pathological variations in the main organs for the curcumin containing TP3 treated group which confirmed there was no substantial in vivo toxicity for the curcumin loaded TP3 [41]. The CS NPs coated with zein was developed as an auspicious encapsulation system to be used for delivery of epigallocatechin gallate (EGCG) [42]. Factors affecting nanoparticle preparation were studied including zein/CS weight ratio, zein concentration and EGCG encapsulation percentage. The structural and physicochemical analyses results indicated that the hydrogen bonds and electrostatic interactions were the main forces occurring in

ACCEPTED MANUSCRIPT nanoparticles creation. The transmission electron microscopy (TEM) displayed a spherical morphology along with smooth surface of the nanoparticles. An initial burst EGCG release was observed from the nanoparticles followed by a slow release. The EGCG release from zein/CS nanoparticles (zein/CS NPs) having higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging ability was relatively greater compared with that of nanoparticles without zein coating in 95% ethanol fatty simulant. Therefore, it was pointed out that the EGCG controlled-release from zein/CS and its antioxidant activity in 95% ethanol fatty simulant

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could long-term protect fatty foods against oxidation.

An electrostatic deposition technique was used for the encapsulation of quercetin into

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CS/lecithin polymeric nanocapsules in order to preserve quercetin degradation and to increase

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its biocompatibility [43]. The morphology, size, storage stability, encapsulation efficiency, anti-proliferative and anti-oxidant activities of quercetin containing nanocapsules (Q-NCs)

encapsulation efficiency (71.14%).

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were studied. The spherical homogeneous Q-NPs had small particle size and great The antioxidant activity and storage stability was

enhanced relative to those of native quercetin. The MTT assay and trypan blue exclusion

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assay displayed the inhibitory influence of Q-CPs on HepG2 cells that were similar to that of free quercetin. Hence, these nanocapsules provided systems for both protection and carriage

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of numerous hydrophobic materials in vivo and food products. HepG2 cells were treated with different concentrations (2-10 mg/mL) of NPs, native quercetin and Q-NPs for 24 h and the MTT assay was carried out to estimate their anti-

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proliferative activities. Noticeable anti-proliferative influence was not perceived in Fig. 11 for the NPs even using 10 mg/mL concentration. Both of the native quercetin and Q-NPs induced dose-dependent cytotoxicity to the HepG2 cells. When using 10 mg/mL concentration, the

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cell viability percentages for the Q-NPs and native quercetin were 40.92% and 46.67%, respectively. The cytotoxicity to HepG2 cells was further examined using trypan blue staining. As trypan blue cannot pass the cytoplasm of living cells, the blue color can be an indicator of membrane damage and cellular destruction. In consistent with the MTT assay, blue spots were observed for the quercetin and Q-NPs treated cells (Figs. 11d and 11e) confirming their strong cancer cell killing capability. Furthermore, noticeable morphological differences were not witnessed between the NPs treated cells (Fig. 11c) and the untreated cells (Fig. 11b). It is known that the molecular oxygen (O 2 ) is essential for human life and it is not very reactive but under certain condition, it can transform to the superoxide anion radical (O 2 ) which damages the human body. Thus, removal of superoxide anion radical is one of the most

ACCEPTED MANUSCRIPT efficient defenses in a living body against different diseases. Fig. 11c reveals a concentrationdependent scavenging ability of quercetin and Q-NPs. Using 10 mg/mL, the scavenging capacities of native quercetin and Q-NPs were 21.39 and 31.39%, respectively [43].

2.2. Application of chitosan in gene delivery Gene therapy has shown great potential for the treatment or prevention of various diseases that cannot be treated with conventional methods [44]. In fact, gene therapy is of

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remarkable research attention that is known as an auspicious approach to treat diseases. This method aims to repair or replace the direct origin of genetic diseases through insertion of

viral infections,

cardiovascular and genetic diseases. The

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for the cancer treatment,

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nucleic acid polymers to patient cells and it is anticipated that this will be a proficient strategy

recombinant DNA technology, discovered in the 1970s, was employed as an impressive

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process for gene regulation. It was first attempted in 1990 to incorporate foreign genes to human cells through retroviral mediated gene transduction [45]. This was a successful effort which was then used for nuclear gene transfer in human as a new strategy in biomedicine. So

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far, numerous efforts have been performed to recognize mutations involved in human diseases using therapeutic genes in order to treat diseases. The low stability, toxicity of nucleic acid

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drugs and low-effective transfection to cells led to gene therapy to be only an experimental technique that is presently in preclinical phase. Also, translation of gene therapy was not considerably efficacious due to several problems including unsuccessful targeted gene

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delivery to diseased sites and cells, gene degradation during delivery and quick clearance in circulation [46]. In order to solve these problems, one fruitful solution is to deliver genes using delivery systems such as CS. Efficient gene therapy causes proficient transfection and

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stable expression of foreign genes into the target cells/tissues, which is highly associated to the delivery systems. Consequently, developing effectual gene delivery systems is an important subject in biomedical application of gene therapy. Nowadays, various techniques are applied to introduce nucleic acids including viral and non-viral vectors but viral vectors can cause diverse human infections. Hence, a natural cationic polymer like CS is extensively used as a non-viral vector system in safe and promising nucleic acid delivery. It was found that CS nanofibers could be utilized in controlled drug delivery application. Moreover, graft copolymerized CS revealed valuable properties for the intracellular delivery of genetic agents [47]. Chitosan nanoparticles have appeared as favorable candidates for gene delivery but slow dissociation of the nanoparticles in cytoplasm is a shortcoming in CS usage. The CS-

ACCEPTED MANUSCRIPT mediated gene delivery was done using polyelectrolyte complexes (PECs) of CS and carboxymethyl dextran (CMD) in order to transfer the micro RNA-145 (miR-145) [48]. The optimized nano PEC preparation process and influences of CS molecular weight and CMD to CS molar ratio (CMD:CS) on the physical properties and in vitro efficiency of the nano PECs were investigated. The size of the nano PECs was dependent to the preparation route, CMD:CS ratio and CS molecular weight. As well, the CS molecular weight and CMD:CS ratio influenced the miR-145 PECs stability in presence of heparin. In vitro experiments

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specified diverse gene transfection efficacies of the nano PECs having different compositions.

balance between the dissociation rate and complex stability.

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Thus, these nano PECs vectors have outstanding capacity for gene delivery with a satisfactory

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Fig. 12 exhibits the gel retardation assay results. The plasmid concentration of 10 µg/ml was completely preserved by all of the nano PECs whereas for the CMD:CS ratio of 5 and the

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plasmid concentration of 15 µg/ml, they did not entirely retain the gene. Also, the nano PECs kept the gene (10 µg/ml) in heparin presence which established the stability of the miR-145 PECs against biological polyanions. However, increasing the DNA concentration led to the

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release of the DNA smear from miR-145 PECs of CS9 (CMD:CS ratio=1) on the gel after incubation with heparin [48].

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As the plasmid expresses both GFP and miR-145, the GFP positive cells were assessed by flowcytometery analysis and fluorescent microscopy in order to determine the gene delivery efficiency using the PECs. Fig. 13A exhibits that the GFP was expressed in MCF-7

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cells after transfection by the miR-145 PECs. The most GFP amounts was expressed in the cells incubated with the miR-145 PECs of CS45 having the CMD:CS ratio of 5. Also, the GFP expression was noticeable in the cells transfected with the miR-145 PECs of CS18

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(CMD:CS ratio=1). Fig. 13B displays the GFP quantity in positive cells after transfection using the diverse PEC formulations. It seemed that GFP was expressed in all samples however the most amount of GFP positive cells was measured following transfection by the nano PECs of CS45 and CS18 having the CMD:CS ratios of 1 and 5. Substantial differences were not detected between the samples transfected with the nano PECs of CS9 with the different CMD:CS ratios but an obvious increase in GFP expression was occurred by increasing the CMD:CS ratio of CS45 PECs. Although it was not a great difference among the nano PECs of CS18 with CMD:CS ratios of 1 and 5, they caused higher GFP expression in the cells compared to that of the nano PECs having a CMD:CS ratio of 0.2. Fig. 14 reveals that the Cy5 PECs uptake was suitable demonstrating the appropriateness of the system for the drug/gene delivery [48].

ACCEPTED MANUSCRIPT Prostate cancer is the most common malignancy in men. Also, CS is of great attention as a suitable biopolymer to encapsulate small interfering RNA (siRNA) due to its cationic nature which can proficiently form nanoparticles containing encapsulated siRNA molecules. Besides, the biocompatibility and biodegradability of CS have led to its application in the in vivo delivery of siRNAs therapeutics. It was reported that CS-carboxymethyl dextran (CMD) nanoparticles efficiently encapsulated the anticancer drugs SN38 and Snail-specific siRNA [49]. Physicochemical, growth inhibition and anti-migration characteristics of the co-delivery

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SN38-Snail siRNA CMD-CS nanoparticles were examined in prostate cancer cells. It was found that in the CSNP-CMD-SN38-siRNA treated cells, the mRNA level of snail was

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reduced from 1.00 to 0.30 (±0.14) and 0.09 (±0.04) after 24h and 48h, respectively. The

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folding induction of E-cadherin and Claudin-1 was increased from 1.00 to 3.12 (±0.62), 3.02 (±0.28) after 24h and 5.6 (±0.91), 4.42 (±0.51) after 48h, respectively. As well, dual delivery

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of SN38 and Snail-specific siRNA by an appropriate nanocerrier (CS NPs) declined the viability, proliferation and migration of PC-3 cells. Thus, the encapsulated SN38 and Snailspecific siRNA in CSNPs can be used as a promising anticancer drug delivery system to treat

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the prostate cancer.

The modified CS was applied as a potential vector in gene delivery to gonadotropin-

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releasing hormone receptor (GnRHR)-expressing cells [50]. This gene carrier is especially valuable for gene therapy to cancers associated to the reproductive system, gene disorders of sexual development and fertility and contraception control. For this purpose, a decapeptide

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GnRH was effectively conjugated to CS and characterized by proton nuclear magnetic resonance spectroscopy (1 H NMR) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized compounds, GnRH-conjugated chitosan (GnRH-

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CS), was capable of condensing DNA to produce positively charged nanoparticles and specially to deliver the plasmid DNA into targeted cells in both three-dimensional (3D) and two-dimensional (2D) cell culture systems. Notably, GnRH-CS displayed higher transfection efficiency relative to unmodified CS. Hence, GnRH-conjugated CS was considered as a favorable carrier for application in targeted DNA carriage to GnRHR-expressing cells. The cell viability of PEI treated cells was considerably declined by increasing the concentration (Fig. 15a) which was related to its high cytotoxicity. However, cytotoxicity was not seen using the concentration ranges of unmodified CS or GnRH-conjugated CS. For both of the GnRH-CS1 and GnRH-CS2, similar results were obtained. Fig. 15b displays numerous dead cells (red fluorescence) in cells treated by 200 µg/ml of PEI polymer relative to the

ACCEPTED MANUSCRIPT normal viable cells (green fluorescence) treated by the same concentration of unmodified CS or GnRH-CS [50]. The targeting ability of the GnRH-CS/pDNA complex was examined in order to approve that transfection was specific and facilitated by interactions between the GnRH ligand and GnRH receptor. Targeted gene delivery to transiently transfected HEK293T which expressed GnRHR (targeted cells) and non-transfected HEK293T (non-targeted cells lacking GnRHR) using the GnRH-CS was compared to unmodified CS obtained by increasing weight

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ratios of polymers and pDNA transporting a GFP reporter gene. Fig. 16a exhibits that substantial differences were not seen in GFP expression by the targeted and non-targeted cells

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treated with unmodified CS/pDNA complex at any weight ratios signifying incapability of

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unmodified CS to distinguish between targeted and non-targeted cells. Nevertheles, GnRHCS could specifically deliver a GFP gene to the targeted cells. Using GnRH-CS/pDNA

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complexes for the cell transfection, GFP expression was completely occurred in targeted cells. GFP expression was not happened in non-targeted cells at any weight ratios, see Fig. 16a. The specificity of GnRH-CS/pDNA complexes was assessed in a 3D multicellular

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spheroid along with 2D monolayer cultures. Comparable results were attained in both 2D and 3D cell cultures (see Fig. 16b). These results prove that gene delivery using the GnRH-CS is

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specific, targeted and dependent on the GnRH receptor [50]. Chitosan microparticles (CSM) can be achieved to be used in delivery of several intracellular payloads. Recently, the effect of cell culture conditions on CSM size and the

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influence of CS on CD59 expression in primary human smooth muscle cells were examined [15]. It was revealed that particle concentration and incubation time in biological buffers increased particle size. The CSM size was suddenly augmented when pH was adjusted

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between 7.0 and 7.5. A CSM loaded by a plasmid with a gene for CD59 (pCSM) was used to transfect cells. The CD59 mRNA and the number of CD59-positive cells were both enhanced subsequent to the pCSM treatment. Surprisingly, CSM enlarged the number of CD59-positive cells. The CS alone increased CD59 expression more effectively than both CSM and pCSM. Therefore, it was confirmed that CS was really bioactive and using the CS alone as control proved that the CS as the delivery systems had activity which was different to that of the payload. As there are still two drawbacks in human gene therapy including lack of biosafety and unsatisfactory delivery efficacy of gene-carriers, very biocompatible CS functionalized Prussian blue (PB) nanoparticles (designated as CS-PB NPs) was produced for photocontrollable gene delivery [51]. The positive charge, ultra-small size (∼3 nm) and great

ACCEPTED MANUSCRIPT physiological stability of CS-PB nanoparticles made it appropriate as a nonviral vector. Furthermore, the CS-PB nanoparticles efficiently converted the near infrared (NIR) light to heat as a result of their strong absorption in the NIR area, which aided the nanoparticles uptake by cells. Under the NIR light radiation, the CS-PB NPs exhibited higher gene transfection efficacy that was much greater than that of free polyethylenimine. All of in vitro and in vivo tests established that the CS-PB NPs had outstanding biocompatibility. Consequently, the CS-PB could be used as a photo-controllable nano-vector for joint gene and

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photo-thermal therapy.

Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) through

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carbodiimide crosslinking were produced as effective gene delivery carriers [52]. The in vitro

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transfection efficacy CUA polyplex was evaluated using 3T3 and HeLa cells. The DNA loading efficiency was measured for the CUA complexes using diverse N:P ratios (1, 2, 4, 6,

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8, and 10). The DNA loading efficiency was calculated equal to >85% for CS, CUA10 and CUA30% and the DNA protection capability of CUA10 and CUA30 nanoparticles was verified by incubation with HindIII and NheI. The cell viability and cell toxicity data

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confirmed the non-toxic nature of CUA10 and CUA30 nanoparticles. The in vitro transfection potency of CUA10 and CUA30 polyplexes was examined for EGFP expression in HeLa and

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3T3 cells and a relative maximum transfection (approximately 10%) was acquired using CUA10 and CUA30 after 96 h transfection. Also, the biocompatibility and viability of CUA gene carrier in transgenic chickens was validated. The in vitro transfection and in vivo

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embryonic viability tests additionally established that the CUA was a promising gene carrier due to its enhanced biocompatibility and DNA protection capacity. With the aim of increasing gene transfer effectiveness and expression stability that are

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significant factors in a fruitful gene therapy, a combined system have was developed for gene transfer which combined the well-recognized non-viral CS polymeric vector with the characteristics of phiC31-integrase that promoted a moderately non-immunogenic, sitespecific integration, with constant gene expression [53]. Also, to solve one of the main challenges in adeno-associated virus mediated gene transfer (the delivery of large genes), the capability of the prepared non-viral vectors were examined by incorporation of a large (8 Kb) transgene.

Polyplexes

were

completely

characterized

for their surface charge,

size,

morphology, pDNA complexation, transgene expression and transfection effectiveness in vitro by means of HEK293 cells. Co-transfection with integrase was accomplished through complexation in a single polyplex or using two distinct polyplex compounds. Transgene expression of CEP290 and GFP that were 8 and 1Kb, respectively, was assessed by flow

ACCEPTED MANUSCRIPT cytometry, fluorescence microscopy and Western blot analysis. The DNA complexation proficiency, particle size and morphology were in agreement with gene delivery in all formulations. Conversely, transfection effectiveness and transgene expression were changed with polyplex and polymer size. Subsequent to delivery by means of CS polyplexes, great levels of GFP expression were still observed after 16 weeks post-transfection. The overexpression of the large transgene was distinguished at least 6 weeks post-transfection. Polyplexes containing phiC1 integrase established sustained gene expression for both of large

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(CEP290, 8 Kb) and small (GFP, 1 Kb) genes. Thus, such a combined approach using integrase and polymer could overcome the size drawback existing in commonly applied

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adeno-associated virus mediated gene transfer methods, in addition to preserving high safety

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with sustained and prolonged gene expression which led to obtain a suitable candidate for gene delivery [53].

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In another research, a nanocarrier system was developed for non-invasive delivery to brain using molecules that were beneficial for gene therapy [54]. Manganese-containing nanoparticles (mNPs) transporting anti-eGFP siRNA were examined in culture of eGFP-

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expressing NIH3T3 cells of mouse fibroblast. After that, the optimum mNPs were tested in vivo in mice. Subsequent to intranasal instillation, mNPs were observed by 7T MRI all over

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brain at 24 and 48 h. the mNPs were active in considerably reducing GFP mRNA expression in Tg GFP+ mice in olfactory bulb, hippocampus, striatum and cortex. Intranasal instillation of mNPS incorporated with dsDNA encoding RFP caused the RFP expression in multiple

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brain sections. The mNPs transporting siRNA or dsDNA were able to deliver the payload from nose to brain. Consequently, this methodology for delivery of gene therapeutics to human would have a significant influence on decreasing of neurodegenerative maladies.

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In vivo assays confirmed the mNPs accumulation in olfactory bulb along with other brain regions upon intranasal installation. The magnetic resonance imaging of anesthetized mice 24 and 48 h following intranasal injection of mNPs that carried anti-GFP siRNA displayed NPs accumulation in several brain regions which was specified by enhanced manganese (Mn) signal intensity in T1 -weighted images that was quantified in four brain regions using parcellation software (Fig. 17). All of the analyzed regions including hippocampus, olfactory bulb, corpus striatum and cerebral cortex demonstrated substantial increase in the Mn signal with peaking at 24 h, Figs. 17E–17F. It was likely that the peak Mn signal was sharper in olfactory bulb if scanning was done before 24 h. The highest variation in the Mn signal was observed in the cerebral cortex, where the signal was enhanced to a mean of 97±17% of baseline at 24 h and decreased to 48.6±17.4% after 48 h. In other regions,

ACCEPTED MANUSCRIPT the peak variations in Mn signals were rather lower however they were similar to the cerebral cortex (hippocampus=89.1±19.7%, olfactory bulb=68.6±19.4% and striatum=77.3±14.4%). All of four brain regions revealed peak Mn signal at 24 h which was followed by a decrease after 48 h [54]. In order to find the extent of gene expression in several brain regions after the intranasal administration, dsDNA encoding RFP was packaged to mNPs. After 48 h intranasal instillation, the RFP was observed in olfactory bulb, cortex, striatum and hippocampus (see

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Fig. 18). The fluorescence signal related to the RFP expressed gene was co-expressed in tyrosine hydroxylase (TH+) neurons in the zona glomerulosa of the olfactory bulb, Fig. 18A.

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The RFP was also expressed in other cell types which are not recognized. In the ventral

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striatum, the RFP co-expression of in neurons (NeuN+cells) indicated the perinuclear pattern of its expression (Fig. 18C). Striatal RFP DNA levels were greater than those of other brain

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regions after 48 h of the nanoparticles intranasal administration (Fig. 18D) [54].

2.3. Application of chitosan in cell/virus/phage/bacteria encapsulation

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Encapsulation is enveloping active agents or core materials in a coating using the embedding ability of a polymeric matrix and recently it has attracted extensive attraction [55].

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The advantageous biodegradable, nontoxic and non-immunogenic polymers can be used to encapsulate, protect and improve the biocompatibility of bioactive components. Numerous efficient encapsulation methods have so far been reported including chelation, microemulsion,

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liposomes and hydrogels. Chitosan is a polycationic biopolymer that can form polyelectrolyte complexes with oppositely charged macromolecules through intermolecular electrostatic deposition. It has been employed as a coating material to encapsulate various bioactive

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agents. For instance, the encapsulated CS materials are used in food industry. In fact, by increasing the healthcare cost, there is a growing consumer demand for functional foods that are food products fortified with some ingredients possessing positive influences on human body such as improving the overall body conditions (e.g., probiotics and vitamins) and modifying risk factors for cancer, coronary heart disease, obesity, osteoporosis, type 2 diabetes and periodontal disease [56]. It is known that stiffness or elasticity of a substrate can affect the phenotypic and functional properties of chondrocytes [57]. Recently, the influence of changing stiffness characteristic of a two-component injectable hydrogel fabricated using CS and oxidized hyaluronic acid (OHA) was examined on the functionality and growth of encapsulated chondrocytes [57]. For this purpose, three diverse gel ratios were obtained including 10:1,

ACCEPTED MANUSCRIPT 10:3 and 10:5 CS-OHA. The stiffness values of the gels were estimated from the force displacement curves by means of atomic force microscopy (AFM). Rabbit articular chondrocytes were harvested and the cells gathered from Passage 2 to 4 were utilized for the encapsulation. The viability and ECM creation of encapsulated chondrocytes were measured at days 7, 14 and 28 post cultures. It was found that when the ratio of hyaluronic acid dialdehyde component was enhanced, the gel stiffness was improved from 130.78±19.83 to 181.47±19.77 kPa which was further verified by the decline in gelling time. Moreover, an

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increase was seen in the number of viable encapsulated cells that preserved their spherical phenotype in less stiff gels but ECM markers (collagen type II and glycosaminoglycans)

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expression was decreased relative to that of stiffer gels. Therefore, it was concluded that the

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gel stiffness considerably affected the chondrocyte microenvironment to preserve their phenotypic integrity and ECM fabrication.

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The effect of hydrogels with different stiffness values on chondrocyte encapsulation was examined using rabbit articular chondrocytes encapsulated in the gels and cultured for 7 days. Live-dead imaging was accomplished to establish the viability and adhesion of cells in

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the gels (Fig. 19). It was observed in the live-dead images that the chondrocytes cultured in the less stiff 10:1 CS-HDA gel exhibited a spherical morphology but the two other

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compositions displayed more cell spreading and a flat morphology at day 7. Also, more dense and uniform cell distribution was seen in the less stiff gel having more viable cells (live cells have green color) compared to the stiffer gels at day 7. Nevertheless, the chondrocytes were

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aggregated having a spherical morphology as the culture time was enhanced to 14 and 28 days. Comparable spherical cell aggregated were found for the 10:1 and 10:3 CS-HDA scaffolds at day 14 that was increased at day 28. The stiffer gel 10:5 CS-HDA revealed

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spherical cellular aggregates which were more spread at day 28 with suitable cell viability. This was attributed to the increased hyaluronic acid amount in the stiffer gels which is a main factor to promote cartilage homeostasis and cell proliferation [57]. The immunostaining of Collagen type II, the characteristic ECM marker of functional chondrocytes and the negative ECM marker collagen type I on the chondrocyte encapsulated hydrogels were accomplished and it was shown that at 7 day culture, the hydrogels 10:3 CSHDA and 10:5 CS-HDA illustrated a greater formation of Collagen type II compared to the less stiff gel (10:1 CS-HDA) (Fig. 20a). This was related to the the stiffness or Young’s modulus of the stiffer gels that was close to those of the native cartilage of rabbits (0.2–0.9 MPa). Such results were supported by the histological collagen staining on the sections of the gels having various stiffness values, see Fig. 21a. In all the three gels, Collagen type I was not

ACCEPTED MANUSCRIPT expressed upon immunostaining which specified formation of more hyaline like cartilage (Fig. 20b). the glycosaminoglycans were stained by Alcian blue on cryo sections of the three gels (Fig. 21b) [57]. In another work, emulsion crosslinking process was employed to achieve CS-genipin microgels which were used as injectable microporous scaffolds [58]. The microgels were characterized by swelling test, light scattering analysis and rheometry of densely-packed microgel solutions. It was indicated that once CS became highly deprotonated above the pK a,

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repulsive forces were decreased and intermolecular attractions led to aggregation of pHresponsive chains which caused microgel–microgel aggregation. The microgel made using the

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most CS and least cross-linker contents revealed maximum yield stress and a storage modulus

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of 16 kPa when it was condensed at pH=7.4. Two oppositely-charged growth factors were encapsulated in the microgels and endothelial cells in order to proliferate as three-dimensional

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microgel scaffolds. Thus, such CS microgels could be used as injectable microporous scaffolds in regenerative medicine.

Several carboxymethyl cellulose-CS (CMC-CS hybrid micro- and macroparticles were

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produced in aqueous medium either through dropwise addition or by nozzle-spray method [59]. These systems were either chemically or physically crosslinked by means of genipin as the reticulation material. The macroparticles (~2 mm) principally showed core-shell structures

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but the microparticles (~5 μm) were actually homogeneous. The crosslinked particles were thermally resistant and robust with low sensitivity to pH variations. Conversely, the physical

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systems were pH-sensitive and presented a notable swelling at pH=7.4 whereas low swelling was witnessed at pH=2.4. Besides, model probiotic bacterium (Lactobacillus rhamnosus GG) was encapsulated in the CMC-CS based particles having suitable viability count. Thus, such

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systems were promising candidates for probiotic encapsulation and effective delivery in the intestinal tract to modulate gut microbiota and improve human health. Currently, researches on phages employed to decrease pathogenic bacteria have raised great attraction but they likely lose their bioactivity in food by the existence of acidic materials, evaporate compounds and enzymes [60]. In order to increase the stability of phages, a CS edible film incorporated with liposome-encapsulated phage was produced [60]. The properties of liposome-encapsulated phage and the CS film loaded by liposomeencapsulated phage were examined. The encapsulation efficiency of phages in liposome was 57.66±0.12%. As well, appropriate physical characteristics were achieved for the CS film. The CS

film containing liposome-encapsulated phage demonstrated great antibacterial

potency against Escherichia coli O157:H7 without affecting the physical properties of beef.

ACCEPTED MANUSCRIPT Therefore, CS film embedded with liposome-encapsulated phage could be used as a favorable antibacterial packaging material for beef preservation. In another work, maximizing efficiency of inactivated avian influenza vaccine was performed by means of safe adjuvants [61]. Chitosan nanoparticles, having an average size of 150 nm and zeta potential of 11.5 mV, were obtained using ionic gelation process. After encapsulation of avian influenza vaccine, their average size was increased to 397 nm and zeta potential was decreased to 4.29 mV. The utmost hemagglutination inhibition (HI) antibody

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titers were revealed in chicken group vaccinated by inactivated avian influenza virus (AIV)CS and then the group vaccinated by inactivated AIV-CS nanoparticles followed by the group

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vaccinated using oil inactivated AIV vaccine, using chicken antigen at two weeks after second

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vaccination. When using duck antigen, the maximum HI antibody titers were observed for the chicken group vaccinated with inactivated AIV oil emulsion vaccine, then chicken group

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vaccinated with AIV-CS nanoparticles, followed by the group vaccinated with AIV–CS. Chicken in the group vaccinated with AIV-CS nanoparticles illustrated the most appropriate data for the lymphocyte proliferation. The phagocytic activity percentage and phagocytic

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index of AIV-CS nanoparticles and AIV–CS groups after three days post first vaccination were considerably enhanced compared to other groups but at 14 days post first vaccination,

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group vaccinated with AIV-CS nanoparticles presented substantial proliferation in phagocytic index and phagocytic activity [61].

Adipose-derived stem cells (ASCs) can potentially treat ischemic diseases but poor

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delivery methods bring about low cellular survival or cells dispersal from target sites. The ASCs contain some angiogenic growth factors that can be used to treat ischemic tissue [62]. Nonetheless, transplantation of dissociated ASCs normally results in quick cell death.

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Consequently, it was intended to prepare a thermosensitive CS/gelatin hydrogel that was able to ASC therapeutic angiogenesis release in a sustained manner [62]. The viability of the encapsulated ASCs was greatly improved after blending gelatin in the CS thermosensitive hydrogel. The steady gelatin degradation during in vitro culturing caused sustained ASCs release from the CS-gelatin hydrogel. The in vitro wound healing assay demonstrated noticeably faster cell migration through co-culturing fibroblasts with ASCs encapsulated in CS-gelatin hydrogel relative to pure CS hydrogel. As well, very greater concentrations of vascular endothelial growth factor were measured in the supernatant of ASC-encapsulated CS-gelatin hydrogels. Co-culturing SVEC4-10 endothelial cells with ASC-encapsulated CSgelatin hydrogels led to substantially greater tube-like structures which designated the hydrogel capability for the angiogenesis promotion. Chick embryo chorioallantoic membrane

ACCEPTED MANUSCRIPT assay and mice wound healing model proved much higher capillary density subsequent to application of ASC-encapsulated CS-gelatin hydrogel. Relative to ASC alone or ASCencapsulated CS hydrogel, extra ASCs were measured in the wound tissue after five days post-wounding

using

ASC-encapsulated

CS-gelatin

hydrogel.

Hence,

CS-gelatin

thermosensitive hydrogels not only preserved ASC survival, but they also supported sustained ASCs release for therapeutic angiogenesis application, thus revealed high clinical promise for the treatment of ischemic diseases.

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ASCs labeled by green fluorescent dye were encapsulated in hydrogels and located at the periphery layer of Hs68 fibroblasts labeled with a red fluorescent dye. Then, the

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fibroblasts were examined by the in vitro wound healing test through scratching the confluent

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cells using a pipette tip (Fig. 22A). The cells images at the hydrogel border and the scratched wound were analyzed after 24 and 48 h. Significantly greater ASCs was released from the

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CS/gelatin hydrogel compared to the CS hydrogel (53.0±5.3 ASCs per power field versus 13.3±2.9 at 24 h with p<0.001 and 61.3±7.4 ASCs per power field versus 19.3±6.1 at 48 h with p=0.002, Fig. 22B). Similar to the in vitro scratched wound model, the migrated

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fibroblasts covered a considerably bigger wound area (17.5±1.3%) in the ASC-encapsulated CS/gelatin hydrogel compared to other groups at 24 h. The artificially generated in vitro

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wound was entirely healed after 48 h using the ASC-encapsulated CS/gelatin hydrogel but cell-free zones were still seen in other groups (Fig. 22C). Enclosed by ASC-encapsulated hydrogels, the endothelial cells started to create a

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vascular network in 4 h (Fig. 23A). The in vitro tube generation by the endothelial cells was quantified through counting the number of branch points per power field. Much more branch points were counted when ASCs were encapsulated to the hydrogels (p<0.005) compared to

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hydrogels alone. The difference between ASC-encapsulated CS/gelatin hydrogel and ASCencapsulated CS hydrogel tended to be significant (19.0±2.0 versus 14.8±1.5 branch points per power field, p=0.082, Fig. 23B) [62]. The capillary formation on chorioallantoic membrane (CAM) was studied to examine the angiogenesis ability of ASC-encapsulated hydrogels (Fig. 24A). Following incubation by CS or CS/gelatin hydrogels alone, the measured capillary areas on CAMs were 6.6±1.0% and 6.9±1.6% respectively. Addition of the ASC-encapsulated CS hydrogel did not significantly affect the increased capillary area on CAM (9.1±1.2%) but the ASC-encapsulated CS/gelatin hydrogel exhibited a considerably higher capillary area on CAM (14.0±2.0%, p<0.01 relative to other groups, Fig. 24B). Also, adding ASC-encapsulated CS/gelatin hydrogel on CAM provided significantly more blood vessel branch points that were 152.5±23.3 branch points

ACCEPTED MANUSCRIPT per power field, p<0.05 compared to 64.7±17.6 for CS hydrogel, 51.7±21.1 for CS/gelatin hydrogel and 99.8±18.7 for ASC-encapsulated CS hydrogel, Fig. 24C. A murine model of cutaneous wound healing was used in order to assess the angiogenic ability of ASC-encapsulated hydrogels in vivo. Immunofluorescent staining of HNA and endothelial lineage-specific marker CD31 was accomplished in wound sections that were harvested

at

day

5

after wound

formation.

Co-localization of HNA and

CD31

immunofluorescence revealed the differentiation of transplanted ASCs to the endothelial

PT

lineage. The HNA immunofluorescence was not seen in wounds treated using the hydrogel alone (Fig. 25A). Treatment by the ASC-encapsulated CS/gelatin hydrogel led to generation

RI

of considerably more HNA+cells in the wound area on post-wounding day 5 relative to those

SC

treated with ASC-encapsulated chitosan hydrogel (131±12 vs. 81±16 cells per power field, p=0.009) or those only treated by ASCs (131±12 versus 49±10 cells per power field, p=0.001;

NU

Fig. 25B). The wounds contained ASC-encapsulated CS/gelatin hydrogel displayed a significantly greater area of CD31+cells compared to the hydrogel alone (11.0±3.2% versus 2.5±0.9% of power field, p=0.003) or the ASC alone (11.0±3.2% versus 2.7±1.2% of power

MA

field, p = 0.003). The ASC encapsulated CS hydrogel showed a significant difference with the hydrogel only (7.3±0.6% vs. 2.5±0.9% of power field, p=0.06) or the ASC only groups

ED

(7.3±0.6% versus 2.7±1.2% of power field with p=0.07, Fig. 25C) [62]. Xanthan-CS hydrogels were prepared and rheologically characterized in the simulated gastrointestinal conditions and then they were employed for encapsulation of anaerobic

EP T

bacterial Bifidobacterium BB01 existing in yogurt [63]. The ability such system (xanthan-CSxanthan) was examined on the viability of B. bifidum BB01 in yogurt during 21 days storage at 4 and 25 ºC. In this system, CS was used as the inner layer in order to coat the

AC C

xanthanprobiotic and xanthan was applied as the outer layer to coat CS-xanthan-probiotic particles. Also, the probiotic survival was explored in bile salt solution and the simulated gastrointestinal conditions. It was pointed out that xanthan-CS-xanthan microcapsules (XCX) and xanthan-CS microcapsules (XC) highly enhanced the cell survival of B. BB01 in yogurt during 21 days storage (at 4 and 25 ºC) compared to free cells. All of the microcapsules displayed greater probiotic cell survival in bile salt solution and simulated gastric fluid relative to that of free cells. The XC microcapsules demonstrated improved release profiles than XCX microcapsules in the simulated intestinal fluid. Thence, it was recommended that the XCX and XC encapsulation systems could effectively be used to enhance bacterial survival both in the gastrointestinal condition and during the yogurt storage.

ACCEPTED MANUSCRIPT 2.4. Application of chitosan in binding to protein drugs Proteins and peptides having health improving features and therapeutic activities have attracted considerable attention. Various health-inducing functions of such bioactive agents include

anticancer,

anti-lipidaemic,

antidiabetic,

antibacterial

and

mineral

binding

characteristics. Protein drugs with biomedical anticancer and antibiotic activities are known as promising therapeutics [64]. The protein drugs are low bioavailable, colloidal instable due to aggregation and greatly susceptible to cleavage by proteases. Besides, because of instability

PT

and short half-life of protein drugs in plasma, patients need numerous injections in order to maintain effective concentration in the therapeutic window. Hence, sustained release of

RI

protein and peptide drugs is a solution to decrease painful and frequent injections. Up to now,

SC

several controlled-release delivery systems have been examined like microspheres, liposomes, nanoparticles and cross-linked hydrogels as systems to enhance the protein and peptide

NU

delivery [65]. Chitosan encapsulation systems are known as one of the most favorable matrices for protein/peptide delivery that is related to its permeation-enhancing influence of CS.

MA

The CS-bioactive glass (BG) composites have been prepared as bioactive coatings for orthopedic applications [66]. It was shown that the bioactivity was increased as a result of the

ED

induced calcium-phosphate/hydroxyapatite creation on the surface when the coating was degraded. In a recent research, protein adsorption and its effect was examined on calciumphosphate precipitation over such composite coatings. For this purpose, 316L stainless steel

EP T

substrates were coated with CS and CS-BG and the coated samples were immersed in two diverse bovine serum albumin (BSA) containing solutions, i.e. H2 O (pH~7.2) and simulated body fluid (SBF). Also, samples were immersed in H2 O and in SBF without BSA to explore

AC C

the impact of protein adsorption on calcium-phosphate precipitation. The surface analysis verified that the BSA adsorption took place on all samples and the protein adsorption was affected by the existence of Ca2+ and PO 4 3− ions. Moreover, the bioactivity as the hydroxyapatite pre-stage formation was considerably enhanced on CS-BG composite coating compared to the bare stainless steel surface. Nonetheless, calcium-phosphate precipitation in SBF was diminished due to the BSA presence [66]. Chitosan/tripolyphosphate (TPP) micro- and nanogels are extensively used as vehicles to deliver protein drugs and vaccines [67]. It is known that the protein uptake by such particles is improved through stronger protein/particle binding but factors controlling their uptake amount (including CS, TPP and protein concentrations) have not been fully investigated. Hence, it was indicated that some differences in the association efficacies (AE-

ACCEPTED MANUSCRIPT values) for protein uptake probably revealed the mainly ignored variations in the particle yield (XAgg), which was defined as the added CS fraction that was self-assembled as particles and (similar to the AE) changed with the CS, TPP and protein concentrations. At first, factors affected XAgg were systematically discovered. Then, it was pointed out that the AE was scaled nearly linearly with the XAgg (which was increased by the TPP and protein-to-CS ratios) until all CS was aggregated as particles. The data collected for different TPP and

PT

protein concentrations were presented as a single AE  XAgg curve for individual protein type. Additional analysis of protein/particle binding illustrated the increase in AE with XAgg which indicated an enhancement in binding sites within the particles and a decline in soluble

RI

(non-particulate) CS molecules which produced soluble protein/CS complexes and competed

SC

with the CS/TPP particles for the unassociated protein. Therefore, it was found that analyzing the various parameters affecting the CS/TPP particle yields can be used to optimize the

NU

protein uptake [67].

An interferometric fiber sensor was experimentally fabricated for detection of hexahistidine tagged microcin (His-MccS) [68]. Such intermodal fiber sensor was prepared using a

MA

no-core fiber functionalized by a CS-nickel (Ni) film in order to directly detect microcin small peptide. The fiber intermodal sensor worked based on the refractive index variations because

ED

selective adsorption was happened at the CS-Ni film. Due to the strong affinity existing between Ni2+ ions and histidine, immobilized Ni2+ ions in the CS film acted as binding sites for the straight detection of hexa-histidine tagged microcin. A comparative assay was

EP T

performed for sensor evaluation using diverse target sizes including full proteins trypsin, BSA and human serum albumin (HSA) having high histidine amount on their surfaces and HisMccS (peptide, 11.6 kDa). It was found that selectivity was achieved for the His-MccS

AC C

compared to trypsin, HSA and BSA. The most important result was fast detection of small His-MccS biomolecule relative to other standard detection techniques. This sensor exhibited His-MccS detection sensitivity of 0.0308 nm/(ng/ml) in the range of 0–78 ng/ml with a concentration detection limit of 0.8368 ng/mL. Amyloid precursor protein (APP) proteolysis is necessary to produce β-amyloid peptides (Aβ) which form senile plaques in Alzheimer’s disease (AD) brains [69]. The β-site amyloid protein precursor cleaves enzyme 1 (BACE1) which is the rate limiting enzyme in creating Aβ from APP; thus BACE1 inhibition is an interesting approach to discover anti-AD drugs. Also, chitosan oligosaccharides (COS) have exhibited numerous biological activities. Hence, the possible inhibitory influence of COS was examined on both BACE1 expression in HEK293 APPswe cells and BACE1 enzymatic activity in vitro. It was found that the cell

ACCEPTED MANUSCRIPT apoptosis was decreased depending on COS dose (100–500 μg/ml) and effectively suppressed the secretion of both Aβ40 and Aβ42. Additionally, treatment with COS caused a remarkable decline in BACE1 mRNA and protein expression level, eIF2α phosphorylation and BACE1 enzymatic effect. Consequently, it was pointed out that COS improved Aβ-associated neurotoxicity that could be attributed to decreased BACE1 enzymatic expression/activity. As CS-protein conjugates are commonly applied in therapeutic drug delivery, the bindings of CS nanoparticles with trypsin and trypsin inhibitor was studied by thermodynamic

PT

analysis and spectroscopic methods [70]. The thermodynamic parameters exhibited that CSprotein binding was occurred mainly through van der Waals and hydrogen bonding contacts

RI

with trypsin inhibitor which formed a more stable conjugate than trypsin. When the CS size

SC

was increased, a more stable polymer-protein conjugate was obtained. The CS complexation led to more perturbations in trypsin inhibitor structure than trypsin along with decrease in

NU

protein α-helix and main increase in random structure. The negative value of Gibbs free energy (G) specified that protein-CS complexation was spontaneous at room temperature. As a result, the CS nanoparticles could be employed to transport trypsin inhibitor and trypsin.

MA

Exploiting the valuable properties of both polysaccharides and proteins is of great importance due to a combination of these two types of biopolymers yield small emulsion

ED

droplets with favorite physical stability [71]. The effect of CS addition to a BSA solution at diverse pH values was evaluated and the resulting conformational variations were examined by UV–Vis absorption spectra as well as fluorescence spectra. Results displayed that the

EP T

BSA/CS ratio and pH significantly influenced the interaction between CS and BSA. Furthermore, the secondary structure and the microenvironment of BSA were altered by the CS addition. Also, CS quenched the intrinsic BSA fluorescence. Hence, these experimental

industry.

AC C

data provided a theoretical guidance to design other helpful ingredients required in the food

The CS stabilized-albumin nanoparticles study was designed and evaluated as NELL-1 protein carriers (CS-NNPs) [72]. The CS-NNPs were achieved through desolvation process and stabilized using CS by electrostatic interactions. The CS-NNPs were characterized for particle size, surface morphology, surface charge and drug loading efficiency. Fluorescein isothiocyanate-labeled CS was applied to approve the homogeneity of CS coating on the BSA nanoparticles. The NELL-1 bioactivity in CS-NNPs and the release kinetics were examined in vitro. The mean particle size and the surface charge using 0.075, 0.15 and 0.3 wt% of CS, respectively, were equal to 368.663±15.470, 382.881±18.767, 390.480±11.465 nm and +25.03±1.42, +30.27±1.80, +31.03±2.05 mV, respectively. The drug entrapment efficiency

ACCEPTED MANUSCRIPT changed from 87.83 to 89.30%. The CS-NNPs obtained using 0.15 wt% of CS could fruitfully control the NELL-1 release and preserve a sustained release up to eight days. Besides, more than 82.67±8.74% bioactivity of the loaded protein was well-maintained in CS-NNPs. Therefore, it was recommended that CS-NNPs could be used as auspicious protein delivery nanocarriers to preserve both the sustained release kinetics and the bioactivity of released NELL-1. A comprehensive research was done on the interactions of CS nanoparticles (15, 100

PT

and 200 kDa with identical 90% deacetylation degree) and two model proteins (BSA and HAS) in order to correlate the CS molecular weight with its binding affinity to protein [73].

RI

The influence of CS on the protein secondary structure and the effect of protein complexation

SC

on the morphology of CS nanoparticles were conferred. It was revealed that the three CS nanoparticles interacted with BSA to produce CS-BSA complexes, mostly by hydrophobic

NU

contacts and the affinity was changed as 200>100>15 kDa. Nevertheless, HSA-CS complexation was primarily happened through electrostatic interactions and the stability order was 100>200>15 kDa. Besides, the contacts occurred between protein and polymer caused a

MA

partial protein conformational variation via a great decrease in α-helix from 63% (free BSA) to 57% (CS-BSA) and from 57% (free HSA) to 51% (CS-HSA). In conclusion, the TEM

ED

micrographs clearly exhibited that the binding of serum albumins to the CS nanoparticles induced a substantial modification in protein conformation and the polymer shape. The tryptophan fluorescence quenching in protein is can be used to explain the

EP T

parameters of drug–protein binding. BSA contains two tryptophan residues including Trp-212 located in a hydrophobic binding pocket and Trp-134 situated on the protein surface, Fig. 26. HSA includes only one tryptophan residue (Trp-214) buried within the protein molecule (see

AC C

Fig. 26). When the binding happens near such Trp, inherent fluorescence quenching by tryptophan is occurred. Fig. 27 displays the influence of the binding of CS-15, CS-100 and CS-200 kDa on the BSA and HSA fluorescence intensity. Apparently, the HSA and BSA fluorescence intensity is slowly decreased by increasing the CS concentration which can be related to the formation of complexes between CS-15, CS-100 and CS-200 and HAS/BSA [73].

2.5. Application of chitosan in tissue engineering Tissue engineering is related to repairing damaged or diseased tissues and organs through controlling the biological microenvironment. In fact, tissue engineering includes several steps that are cell proliferation, differentiation and synthesis of extracellular matrix

ACCEPTED MANUSCRIPT (ECM) [74]. An ideal tissue engineering scaffold is a template used for three-dimensional (3D) tissue growth in order to mimic the natural tissue microenvironment by offering a porous structure for the tissue growth, oxygen diffusion and nutrients delivery. Also, it can interact with the neighbouring cells and preserve the phenotype of the renewed tissue. A perfect scaffold must be biodegradable, biocompatible and promotes cell adhesion, proliferation as well as retains the metabolic action of cells. Besides, the scaffolds with appropriate pluripotent stem cells, angiogenic effect and prolonged nutrient resource will accomplish the of numerous

tissues

[75].

An

implantable

scaffold

must exhibit high

PT

regeneration

compatibility with body, suitable mechanical properties, morphology, porosity, healing and

RI

tissue replacement ability [75].

SC

As a result of its outstanding characteristics, CS has the capability to create scaffolds with excellent interconnected porosity and favorite shapes like sponges, hydrogels, two-

NU

dimensional sheets/fibers and 3D porous structures to be employed for the enhanced cell viability through supplying sufficient oxygen and nutrients [76]. The CS-based scaffold materials display controlled delivery of loaded therapeutics and growth factors indicating they

MA

are pertinent candidates for regenerative applications and tissue engineering [76]. Thus, CS scaffolds could substitute for damaged or missing tissues to stimulate cell attachment and

leads

to

its

ED

proliferation. As well, the existence of primary amino and hydroxyl groups on the CS chains chemical modification

functionalities and features.

to

achieve

different

derivatives

with

preferred

EP T

Fibrous scaffolds with different ratios of CS to poly (lactic acid) (PLA) were obtained by electrospinning method [77]. Subsequent to crosslinking using the glutaraldehyde vapor, the mechanical properties, structures, hydrophilicity and in-fiber chemical interactions of the

AC C

scaffolds were evaluated. It was exhibited that the fiber diameter was diminished with the CS concentration whereas hydrophilicity and mechanical properties were enhanced. Furthermore, scaffolds with aligned fibers had greater mechanical strengths and biocompatibility compared to scaffolds containing random fibers. Particularly, scaffolds having aligned fibers produced with PLA:CS ratio of 7:1 supported cardiomyocyte viability, prompted cell elongation, and enhanced creation of sarcomeric α-actinin and troponin I. Hence, it was found that composite scaffolds made using PLA-CS fibers had high potential to engineer cardiac tissue and to accelerate the myocardia regeneration. Fig. 28a exhibits that cardiomyocytes grown on random fibers and tissue culture plates have lost structural polarity, round morphology and express α-actinin diffusely. On the other hand, cells that were seeded on aligned PLA/CS nanofibers maintained very polar

ACCEPTED MANUSCRIPT morphology and more plentifully accumulated α-actinin almost parallel to the nanofibers. Particularly, cardiomyocytes grown on aligned fibers with 7:1 PLA:CS more amply express α-actin than other fibers. The elongated morphology and alignment of the cells revealed that an anisotropic tissue having great ability to contract has been formed. Furthermore, Tn-I caused a similar effect, especially the highly parallel Tn-I arrangement is aligned on the A7 fiber having a shuttle-like shape (see Fig. 28b). The PLA/CS fibers afford suitable biological and chemical properties to support cardiomyocytes to deliver more intercellular guidance

PT

[77].

It is well known that CS-based porous structures have widely been investigated around

RI

the world as promising tissue engineering scaffolds [78]. Although there are differences in CS

SC

polymers obtained using squid pens or crustacean shells, with the former is more reactive and simply accessible with a higher degree of deacetylation (DD), in most efforts the crab or

NU

shrimp CS are used due to they are easily obtainable in commercial sources. Hence, in a recent study, high potential of CS materials achieved from squid pens was examined for their biomedical applications

[78].

Using

freeze-dried

scaffolds produced

for soft tissue

MA

engineering, the effect of the DD of CS polymer and the freezing temperature during processing were explored on their performances. In this procedure, CS was attained by

ED

deacetylation of β-chitin earlier isolated from endoskeleton of giant squid Dosidicus gigas (DD=91.2%) and it was compared to a commercially existing batch acquired from crab shells (DD=76.6%). The CS solutions were frozen at −80 or −196 °C and additionally freeze-dried

EP T

to get 3D porous structures. The scaffolds prepared at −196 °C had a compact structure with smaller pores, but those obtained at −80 °C exhibited a lamellar structure with larger pores. The compressive modulus was changed from 0.7 to 8.8 MPa. All scaffolds were stable up to

AC C

four weeks in phosphate buffer saline (PBS) and in the presence of lysozyme. In addition, the squid CS scaffolds processed at −80 °C supported ATDC5 chondrocyte-like cells adhesion and proliferation. Consequently, it was proposed that these squid CS scaffolds could be exploited for application in cartilage tissue engineering. In order to enhance the hydrophilicity of CS fiber, N-carboxyethyl CS fiber was developed by Michael addition between CS fiber and acrylic acid and the structure was characterized by 1 H NMR spectrum [79]. The N-substitution degree, measured from the 1 H NMR, ranged from 0.10 to 0.51 by changing the molar ratio of CS to acrylic acid. Some characteristics of N-carboxyethyl CS fiber such as crystallinity, mechanical and thermal properties and in vitro degradation were explored. It was found that creation the carboxyethyl group onto the CS chain ruined the intra/intermolecular hydrogen bonds which led to

ACCEPTED MANUSCRIPT hydrophilicity enhancement. Indirect cytotoxicity evaluation of carboxyethyl CS fibers was accomplished using L929 cell line indicating the N-carboxyethyl CS fiber was nontoxic to these cells. Hence, the N-carboxyethyl CS fibers could be employed as potential scaffold materials in tissue engineering. Some gelatin-carboxymethyl CS scaffolds were fabricated, characterized and applied in dermal tissue engineering [80]. The influence of carboxymethyl CS and gelatin ratio was assessed on their physical, chemical and biological properties and drug release kinetics. The

PT

scaffolds were produced using freeze drying process and characterized through FTIR and SEM analysis techniques. It was found that the scaffolds were greatly porous with pore size

RI

changing from 90 to 170 μm, highly adsorbed (400–1100%) and retained (>300%) water. The

SC

scaffolds degradation mediated by collagenase depended on the gelatin content in the formulation. Also, a minor but significant alteration was perceived in their biological features.

NU

All of scaffolds stimulated adhesion, spreading, growth and proliferation of 3T3 mouse fibroblasts. As well, the cells seeded onto the scaffolds revealed expression of collagen type I, HIF1α and VEGF, reflecting a sign for their growth and proliferation accompanied by activity

MA

to promote angiogenesis throughout wound healing. Besides, the scaffolds exhibited sustained release of both bovine serum albumin and ampicillin which established their appropriateness

ED

as therapeutic delivery vehicles. Overall, it was recommended that gelatin-carboxymethyl CS scaffolds could be employed as suitable materials in dermal tissue engineering. It has been recognized that electroactive scaffolds fabricated using conductive polymers

EP T

are able to support tissue regeneration and repair; nevertheless, such polymers are nondegradable which cannot be eliminated from body [81]. In order to tackle this drawback of conductive polymers, an injectable electroactive hydrogel including pyrrole oligomers was which showed

AC C

developed

both exceptional properties of electrical conductivity and

biodegradability. The pyrrole oligomers were first synthesized by chemical polymerization, which were amorphous with a non-globular morphology. Next, three diverse compositions of injectable

CS/beta

glycerophosphate

hydrogels

were

synthesized

comprising

various

concentrations of pyrrole oligomers and their morphology, chemical structure, swelling ratio, conductivity, in vitro biodegradation and gelation time were investigated. It was revealed that an increase in oligopyrrole amount diminished the pore size and improved the swelling ratio, gelation time, degradation time and conductivity. Among all of the hydrogels, the sample having

a

pyrrole

oligomer:CS

ratio

of 0.1

(w/w) displayed

the most noticeable

biocompatibility, biodegradability, swelling ratio, electro-activity and pore size. Thus, it was selected as an ideal electroactive hydrogel [81].

ACCEPTED MANUSCRIPT In another research, CS-co-hyaluronic acid cryogels were prepared using glutaraldehyde crosslinker at subzero temperature and used as tissue-engineering scaffolds [82]. The cryogels contained different ratios of CS and hyaluronic acid (0, 10, 20, 30 and 50 wt% hyaluronic acid). The morphological investigation displayed that the macroporous cryogels had 90–95% porosity and 150–200 μm pore size. The biomaterial and mechanical properties of pure CS were particularly enhanced through fabrication of copolymer with hyaluronic acid in diverse concentrations. These cryogels demonstrated biodegradable nature and fast swelling behavior.

PT

It was proved that the swelling ratio, flexibility and durability were increased after copolymerization of hyaluronic acid. The MTT cell viability test established that the cryogel

RI

scaffolds presented the highest ability in the proliferation of both 3T3 and SaOS-2 cells by

SC

increasing the hyaluronic acid content and they had no significant cytotoxicity influences on 3T3 SAOS-2 and fibroblast cells. The SEM micrographs of the cryogels displayed that their

NU

very porous networks were contributed to adhesion and spreading of SaOs-2 and 3T3 cells. Consequently, the CS-hyaluronic acid cryogel scaffolds could be used in both in vitro and in vivo tissue engineering applications [82].

MA

The CS and hyaluronic acid cryogel tissue scaffolds were applied for cell proliferation and growth. The SEM images in Fig. 29 display the seeded 3T3 mouse fibroblast cell and the

ED

SAOS-2 bone cancer (osteosarcoma) cells after 24 h. the attachment efficacies of seeded 3T3 and SAOS-2 cells on scaffolds were observed by SEM micrographs (Fig. 30) indicating growth of cells in the pores and their adherence to scaffolds surfaces. The CS scaffolds limitat

EP T

the survival of cells and cells are migrated to the pores because of its more rigid structure and narrower copolymer pores when comparing CS cryogel scaffolds with CS copolymer scaffolds. For the CS copolymers with hyaluronic acid, the cells simply migrate to the pores

AC C

owing to their large pores and specific flexible structure. Besides, the seeded cells on the scaffold diagnose the hydrophilic nature of CS and hyaluronic acid thus they can well be adhered. The pores permit cells to migrate toward the cryogel matrix. Also, pore size affects the metabolic activity of cells including waste removal and nutrient supply. Nevertheless, the cell viability may be increased by increasing the hyaluronic acid ratio [82]. Several photocrosslinkable water-soluble maleilated CS and methacrylated poly (vinyl alcohol) were synthesized and then maleilated CS-methacrylated poly (vinyl alcohol) (MCSMPVA) hydrogels were achieved by UV irradiation [83]. Some hydrogels properties were evaluated including morphology, rheology, swelling and mechanical characteristics. It was found

that

the

MCS-MPVA

hydrogels

indicated

rapid

gel formation rate (whole

transformation to gel in 150 s), enhanced compressive strength at 0.169±0.011 MPa and fast

ACCEPTED MANUSCRIPT absorbent capability. These features could be adjusted by controlling the weight ratio of MCS to MPVA. Also, the indirect cytotoxicity test established that the photocrosslinked hydrogels were compatible to L929 cells of mouse fibroblasts signifying their ability as tissue engineering scaffolds. Recently,

chitosan-hydroxyapatite

(CS-HA)

nanocomposites

exhibiting

intercalated

structures were fabricated [84]. For this purpose, hydroxyapatite was synthesized by the solgel method and formic acid was used as solvent to get stable dispersions of nano-sized HA

PT

particles in the polymeric solution. The CS-HA dispersions were prepared using of 5, 10 and 20 wt% of HA. Self-assembly of HA nanoparticles throughout drying the films formed

RI

homogeneous CS-HA nanocomposites. The AFM and SEM images verified the existence of

SC

evenly distributed HA nanoparticles in the CS matrix. The XRD patterns and cross-sectional SEM micrographs displayed that layered nanocomposites were achieved. Complete CS

NU

degradation in the thermogravimetric analysis (TGA) resulted in the formation of nanoporous 3D scaffolds comprising hydroxyapatite, calcium pyrophosphate and β-tricalcium phosphate. The CS-HA composites could be known as auspicious materials for application in bone tissue

MA

engineering.

Diverse quantities (0.5, 1 and 2) of resol resin (RS) were added to the CS-

ED

hydroxyapatite (CS-HA) in order to develop tri-constituent CS-HA-RS nanoensembles by a facile co-precipitation process [85]. The TEM, SEM images indicated irregular interconnected rough morphologies with homogenous distribution of needle like particles with average sizes

EP T

changing from 12 to 19 nm. The TGA and mechanical analysis prove that the CS-HA-RS nanocomposites had higher thermal stability and mechanical strength compared to the CS-HA (binary) composite. The CS-HA-1RS nanocomposite exhibited improved protein adsorption

AC C

and alkaline phosphate activity with exceptional apatite formation capability than CS-HA-RS (0.5, 2) and CS-HA nanocomposites. Accordingly, the CS-HA-1RS nanocomposite was chosen to test as a bare implant to repair critical-size calvarium defect (8 mm) in albino rat. The radiological and histopathological tests pointed out that CS-HA-1RS stimulated the bone regeneration as soon as two weeks post-implantation signifying extraordinarily quicker healing of calvarial defect compared to Cerabone. Hence, the CS-HA-1RS could be used as a potential biomaterial in bone tissue engineering. Different steps occurred in surgeries are presented in Fig. 31(i). All animals displayed uneventful wound healing in one week at selected times post-implantation. Post-surgical edema was happened at surgical site of each rat that was vanished during 2–4 days after the procedure. All of twenty-four rats did not reveal any signs of infection. The general health,

ACCEPTED MANUSCRIPT activity, weight and exertion were normal in the entire follow up phases. Bone formation was not observed in time period of 4 weeks and 8 weeks. The only difference noticed between temporal groups was increased collagen fiber (obviously seen in Sirius red [SR] stain) and vascularity at the defect site, Figs. 31 (ii)a, 32a. The biopsy results for the tissues are in well agreement with the X-RAY & RVG data obtained prior to the histopathological processings, Fig. 33(i)a,d–g,j. The radiographic analysis illustrated that the bone defect of the sham control group was 18.20±0.18% and

PT

31.60±1.54% (gain in bone density=GBD) that was repaired at 4 weeks and 8 weeks, respectively, Fig. 33(ii). For the positive control group for which the defect was filled by the

RI

commercial formulation Cerabone (CB group), the complete absence of bone formation was

SC

seen at the end of 4 weeks, Fig. 31(ii)b. The defect area was filled only by irregularly arranged collagen fibers resulting in a loculated shape containing clumps of CB particles

NU

enclosed by a delicate fibrous tissue signifying CB only experiences partial integration, Fig. 33(i)b,h. Nevertheless, the GBD was only 40.81±1.12% [Fig. 33(ii)] that may only be related to the existence of non-dispersed CB at the bone defect site but it was not corresponded to

MA

actual bone creation verified by the histopathological data. However, promising results were achieved using CS-HA-1RS nanocomposite scaffold so that superior bone healing was

ED

noticed in comparison to both the sham control and CB groups in four weeks. Such results were in consistent with the X-ray and RVG analysis data, Fig. 33(i)c,i. Also, the immature lamellar bone was found in the site filled with CS-HA-1RS suggestng its highly strong

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osteoregenerative ability which supported new bone formation as well as rapidly remodeled it into lamellar bone during a very short time of four weeks, Fig. 31(ii)c. The bone formed was very look like to normal mature host bone (this was occurred without previous cell seeding or

AC C

using any growth factors). The whole amount of formed bone in the CS-HA-1RS group was greatly enhanced with GBD (76.04±1.13%) at four weeks, Fig. 33(ii). After 8 weeks and in contrary to the CB group, the CS-HA-1RS group presented more progressive calcification and the defect was almost filled by freshly created mature bone analogous to the host bone, Fig. 32(b,c). The growing free border situated near the defect center was enclosed by well-ordered osteoblasts and random osteoclasts compared to those in the CB group that did not display full sealing of the bone defects confirmed by the H & E as well as the SR stained sections. An extraordinary new bone generation and remodeling were seen about CS-HA-1RS implant along with the ingrowth of recently formed mature lamellar bone without encapsulation of fibrous tissue signifying a higher bone regeneration relative to CB implants. Remarkably, the CB was not totally degraded even after 8 weeks demonstrating

ACCEPTED MANUSCRIPT its very slow dispersion rate (see Fig. 32b). Additionally, the new bone formation zone during 8 weeks in the CS-HA-1RS was considerably bigger than that in CB group which was observed in its radiographs in Fig. 33(i)e,k,f,l giving the GBD values of 92.69±0.66% and 77.69±0.93%, respectively, see Fig. 33(ii). Consequently, in vivo tests obviously point out that

the

CS-HA-1RS

nanocomposite

scaffold

has

improved

osteointegration

and

osteoinduction. The harvested calvaria of three groups including control, CB and CS-HA-1RS after 8

PT

weeks post implantation are observed in Fig. 33i.(m–o). In addition, inflammation or necrosis was not noticed on both CS-HA-1RS and CB biopsy samples signifying the implants were

RI

well tolerated without any evident toxic influence in the neighboring tissues. To exclude any

SC

probability of harmful effects on the vital tissues (kidney and liver) by the systemic absorption of the new scaffold, such tissues were examined by histopathological test and

NU

compared to sham control group, Fig. 32(d–g). The kidneys and livers were harvested after 8 weeks and fixed in Karnovsky fixative for two weeks and finally processed by paraffin embedding. The renal corpuscles and renal tubules in the CS-HA-1RS group were greatly

MA

similar to control group. As well, the hepatocytes and sinusoids in both CS-HA-1RS and control group were very comparable. The kidney parenchyma seemed normal having intact

ED

renal corpuscles without congestion. Renal tubules possessed intact lining and no renal casts. Liver exhibited intact histoarchitecture and normal hepatocytes and sinusoids. Apparent necrosis or congestion was not noticed which verified the nanocomposite scaffold was not

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hepatotoxic or nephrotoxic, Fig. 32(e,g).

Because the CS-HA-1RS nanocomposite scaffold led to lamellar bone formation in four weeks and whole mature bone was created after 8 weeks, it was mandatory to perfoem an

AC C

extra short-term test to display the lowest initial time (latent period) necessary for osteogenesis to start and detect the transition phase of bone formation to further recognize its mechanistic action. Hence, the experiments were done for two weeks using the CS-HA-1RS, Fig. 34(a–e). This test was not replicated for control and CB groups as there was no bone formation even after four weeks. After two weeks post-implantation, initial phase of new bone generation was witnessed at the margin of the calvarial defect evidently indicating the fibrous tissue, woven bone and lamellar bone (new mature bone), Fig. 34a. The in situ bone defect site (top view) and the bone defect site inside (bottom view) are revealed in Fig. 34(b,c). The freshly created lamellar bone had a comparable density to the normal bone which was obvious from its radiographs approving the new bone was formed originally from the

ACCEPTED MANUSCRIPT host bone, grew and extended to the defect center, Fig. 34(d,e). The bone density degree was 61.14±4.15% [85]. Photocrosslinkable hydrogels

obtained

using natural materials are promising for

application as scaffolds in tissue engineering but their drawbacks weak are formation and poor mechanical characteristics [86]. Recently, photo-clickable thiol-ene hydrogels based on CS were synthesized by photopolymerization of maleic chitosan (MCS) and thiol-terminated poly (vinyl alcohol) (TPVA) by means of a biocompatible photoinitiator [260]. It was shown

PT

that the rheological and absorbing properties of the MCS-TPVA hydrogels could be tuned by changing the TPVA amount in the feed. Strong intermolecular hydrogen bonds were formed

RI

between TPVA and MCS. The MCS-TPVA hydrogel (MT-3) displayed a fast gelation (<120

SC

s), enhanced stiffness (G'=∼5500 Pa) and compressive strength (0.285±0.014 MPa) that were vital for the hydrogel scaffolds, mainly for the injectable hydrogel scaffolds. Also, the

NU

photocrosslinked MCS-TPVA hydrogels was cytocompatible which promoted the L929 cells attachment and proliferation confirming they were promising tissue engineering scaffolds. In another work, composite nanofibers were produced by electrospinning method using

MA

magnesium oxide (MgO), poly(ε-caprolactone) (PCL) and CS with diameters ranged as 0.7– 1.3 μm [87]. The physicochemical properties including morphology, mechanical strength and

ED

integrity in aqueous environment were evaluated. The cellular compatibility was examined by cell viability tests and microscopy imaging and it was revealed that the nanofibrous membranes supported

the viability and attachment of 3T3 cells. Accordingly, such

EP T

nanofibrous composites could mimic the physical structure and function of tissue extracellular matrix indicating they could be employed in tissue engineering purposes. Fabrication

and

characterization

of

bioactive

scaffolds

prepared

using

CS,

AC C

carboxymethyl chitosan (CMC) and magnesium gluconate (MgG) were accomplished [88]. The scaffolds were prepared by subsequent freezing promoted phase separation and lyophilizing polyelectrolyte of CS, CMC and MgG complexes. The scaffolds revealed uniform porosity with greatly interconnected pores having sizes in the range of 50-250 mm. The elastic moduli up to 5 MPa and compressive strengths up to 400 kPa were measured. Also, the scaffolds were remained intact, retained their original 3D frameworks under in vitro testing conditions. Such scaffolds did not display cytotoxicity to 3T3 fibroblast and osteoblast cells. Therefore, these scaffolds were efficient and appropriate for tissue engineering. In another work, CS composite scaffolds were developed with controllable internal architectures for bone tissue engineering [89]. The CS based composites were produced through changing montmorillonite (MMT) and HA contents in order to obtain macro-

ACCEPTED MANUSCRIPT spherical 3D scaffolds upon direct agglomeration of the sintered macrospheres. The physicochemical, biological and mechanical properties of the CS, CS-MMT, CS-HA and CSMMT-HA 3D scaffolds were characterized. The reinforcement using HA and MMT caused decreased degradation rate and swelling. Also, compared to pure CS, the CS-HA-MMT composites demonstrated enhanced protein adsorption and hemocompatibility. Sintering of the macrospheres adjusted the swelling capacities of the 3D scaffolds which were much significant to maintain their mechanical strengths. The CS/HA/MMT composite scaffold

PT

exhibited 14 folds enhancement in the compressive strength in comparison to the pure CS scaffold. As well, the scaffolds supported the MG 63 cell proliferation. As a result, it was

RI

concluded that the CS-HA-MMT composite macrospheric 3D scaffolds could find practical

SC

applications in bone tissue regeneration.

NU

2.6. Application of chitosan in preparation of implants

Short-term contact of implant with blood can lead to adsorption of plasma proteins, calcium, platelet and bacterial adhesions on the implant surface. The adhered platelets could

MA

be then activated resulting in the coagulation cascade and after that thrombosis. At long-term, calcification of implants (heart valves, breast implants and stents) may be happened by

ED

growing calcium phosphates or other calcium salts deposits. Moreover, implants suffer from high plaque accumulation which is commonly related to the two most important pathogens, i.e. Porphyromonas gingivalis and Streptococcus mutans [90]. For instance, colonization

EP T

occurs on the fixed appliances in prosthodontic therapy due to biofilm forming bacteria which could destroy the periodontal tissue [90]. Problems including thrombosis, calcification and bacterial growth can interfere with the implant function, decrease its lifetime and bring about

AC C

implant explanation. Therefore, researches on interaction of blood with biomaterials were focused on surface treatment to inhibit calcification and thrombosis [91]. In order to enhance the lifetime (short and long terms) of clinical implants, they should be coated with a biocompatible material as a bio-interface that is able to overcome the above-mentioned challenges. Among various polymers, CS appears as an extremely promising substance due to its versatility and potential to solve these complications [92]. Numerous metal coated implants have up to now been examined against dental pathogens leading to biofilm formation and failure of dental implants [90]. Nanoparticles can be used accompanied by native biomolecules in order to improve the activity of such bioactive compounds. In an effort, the efficacy of Ag conjugated CS nanoparticles was estimated as a prospective coating material for titanium dental implants [93]. The bioactive

ACCEPTED MANUSCRIPT CS was obtained from A. flavus Af09 and conjugated with Ag nanoparticles. The Ag-CS nanoparticle revealed good growth inhibition influence against two key dental pathogens including P. gingivalis and S. mutans. The Ag-CS inhibited the adhesion of these two bacteria and decreased the biofilm formation. Also, the nanoparticle inhibited the quorum sensing production in these bacteria. The naturally extracted CS did not exhibit cell cytotoxicity confirming its biocompatibility. Hence, coating the titanium dental implants by the Ag-CS could be an advantage as the corrosion resistance of dental implants to increase the

PT

passivating property of the implants [93].

Lower biofilm formations were observed upon treatment by gentamicin and CS

RI

compared to those conjugated with Ag which were significantly lower than the positive

SC

control. Images obtained by congored staining evidently exhibited a decrease in biofilm thickness so that a noteworthy reduction in biofilm formation was realized as S mutans was

NU

grown in the existence of CS conjugated with Ag nanoparticle (Fig. 35a). The image analysis using the ImageJ software displayed 60% biomass decrease upon treatment with antibiotic and nanoparticles, Fig. 35b. The treatment of HGF cells did not demonstrate any cytotoxic

MA

effects for the Ag-CS. Also, substantial differences were not observed in the proportion of live cells in AOEB staining (see Fig. 36) [93].

ED

It was attempted to fabricate CS-based hydrogel implants to be used in peripheral nervous tissue regeneration [13]. The fabrication method was based on electrodeposition using a solution of CS and organic acid. The solution was supplemented with hydroxyapatite

EP T

to enhance the mechanical strength of the implant. Also, the hydroxyapatite was acted as a source of calcium ions. The effect of the polymer and the additive concentrations were assessed

on mechanical, chemical and biological characteristics of the implant. The

AC C

physicochemical properties of the prepared structure were highly dependent to the initial solution composition. The in vitro cytotoxic and pro-inflammatory assays displayed the biocompatibility of implants. The directly compressed tablets were developed for implantable delivery of risedronate sodium to treat osteoporosis and compare the mechanism and kinetics of drug release from biodegradable CS and non-degradable polyvinylchloride (PVC) polymer matrices [94]. The compositions and procedure parameters were optimized by a mixed 2 and 3 level full factorial design. Critical Quality Attributes (CQA) were studied including diametral breaking hardness, porosity and drug dissolution speed. It was revealed that there were substantial differences between the behaviors of the two polymers. The CS displayed poor compressibility which caused weak mechanical properties and rapid disintegration of the CS-based tablets.

ACCEPTED MANUSCRIPT However, despite its quick disintegration, the CS-based matrices demonstrated one-week-long continuous drug release, which could be related to strong drug-carrier interactions. The existence of intermolecular hydrogen bonds was established by FT-IR and near infrared (NIR) spectra.

Conversely, the PVC-based composites presented outstanding compressibility,

suitable tablet hardness and little porosity. The tablets stayed intact throughout the dissolution and showed a slower release rate than those of CS-based matrices. The NIR spectra did not illustrate intermolecular interactions which suggested the dissolution rate was happened due

PT

to the porosity of tablets whereas the FT-IR spectra provided some details about the molecular interactions occurred during the drug release mechanism.

RI

Several CS–carbon nanotube implants were developed as tubular hydrogels that were

SC

enriched by calcium ions [95]. The hydrogels were intended to be used in tissue engineering particularly peripheral nervous tissue regeneration. The fabrication method was based on the

NU

electrodeposition which showed important benefits over current approaches so that the implants could quickly be acquired at any requisite dimensions. Therefore, this could be considered as an effective method to treat patients having peripheral nerve injuries. Both

MA

single walled and multiwalled carbon nanotubes improved the mechanical features of the tubular hydrogels. Also, controlled existence of calcium ions added using hydroxyapatite

ED

enhanced the regenerative response. The in vitro cytotoxic tests on mouse cell lines (mHippoE-18 hippocampal and L929 fibroblasts cells) and pro-inflammatory assays on THP1XBlue™ cells exhibited that the implants were biocompatible. Consequently, the immune-

EP T

and nervous-safety of implants could be evaluated on animal models.

2.7. Application of chitosan in preparation of contact lenses

AC C

Drug delivery using ocular therapeutics is a challenge which is due to the anatomical and physiological restrictions of the eye which does not allow achieving correct therapeutic concentration at the desired site of action. Hence, clinicians recommend using numerous dosages, which causes nonconformity by patients and less economical. To tackle these problems, other ocular delivery systems have been explored including ocuserts, in situ gels, liposomes and nanoparticles. A principally attractive form of these delivery systems is contact lenses which are thin and curved plastic disks designed to protect the cornea by clinging to the eye surface through surface tension [96]. Currently, therapeutic ophthalmic lenses have been developed that are able to release suitable drugs in a long time in order to circumvent the ineffective and tedious eye drop administration. In this context, numerous efforts have been done by researchers to improve

ACCEPTED MANUSCRIPT drug delivery using soft contact lenses (SCLs) [97]. Also, in case of cataracts which are one of the most common eye diseases, therapeutic intraocular lenses (IOLs) are fabricated to avoid postoperative infectious problems after surgery [98]. Indeed, one of the foremost complications in the application of drug-loaded ophthalmic lenses to replace the topical usage of eye drops is controlling the drug release. Usually, the drug release from such devices occurs as an early burst release followed by decline in drug release to levels under therapeutic dose. To solve this challenge, some approaches are proposed to ensure a sustained medication

PT

delivery to the eye throughout the requisite time period at a controlled rate. These approaches include addition of chemicals to reversibly interact with the drug, using nanocarriers such as

RI

liposomes, micelles and nanoparticles, incorporation of diffusion barriers to the drugs like

SC

vitamin E aggregates [99]. Another method is the implementation of coatings in commercial lenses whose production procedures and properties have been optimized. Presently, coatings

NU

are employed in commercial SCLs to enhance the surface wettability and lubricity which have led to higher comfort to the users. One of objectives in such coatings is to prevent the adsorption of proteins and microorganisms from the ocular tear fluid in order to evade eye

MA

infections by the users of contact lenses. Various coatings are provided to SCLs that are fundamentally based on polyelectrolyte multilayers achieved by the grafting/adsorption of

ED

particular molecules and immobilization of liposomes at the lens surface [100]. Moreover, natural polymers like CS and alginate can be used for application as coatings in contact lenses.

EP T

In another attempt, an extended wear therapeutic contact lens (TCL) was fabricated by molecular imprinting method for the sustained delivery of timolol maleate (TML) [14]. The designed TCL was composed of a TML imprinted copolymer of carboxymethyl CS-gmethacrylate-g-polyacrylamide

AC C

hydroxyethyl

(CmCS-g-HEMA-g-pAAm)

which

was

embedded to a poly HEMA matrix (pHEMA). The TML reloading to the lens was proved by UV–visible spectra which exhibited the outstanding reloading capacity was 6.53 μg TML/TCL. The in vitro drug release after each cycle in lacrimal fluid was fitted to Higuchi model which proposed the diffusion release mechanism was occurred with no polymer degradation. As well, the TML release kinetics pointed out a sustained drug delivery which could efficiently attain the TML therapeutic index and provided a onetime glaucoma treatment. The biological activity of eluted drug subsequent to each cycle and cell viability of the TCL were substantiated by means of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,3bis(2-methoxynitro-5-sulfophenyl)-5-(phenylaminocarbonyl)-2H-tetrazolium (XTT) assay, respectively.

hydroxide

ACCEPTED MANUSCRIPT Balb/3T3 Clone A31 cell lines are frequently applied to assess cell viability in ophthalmic preparations because they look like the retinal receptors. Cell viability was examined using XTT assay which measured mitochondrial activity. The cell viabilities of the disks were calculated to be 77.5, 74.1, 71.8, 67.2 and 61.0%, respectively for 1.50, 3.00, 6.25, 12.50 and 25.00 µg/mL of TCL. Such values exhibited outstanding cell viability and notably the control and sample displayed nearly the same values (Fig. 37). Thus, results proved the biological approval of the prepared TCL and its practical usefulness [14].

arena

of oculopathy

therapy,

PT

Despite the fact that hydrogel contact lens is of great attention as delivery carrier in the traditional hydrogel does not illustrate

desired

drug

RI

encapsulation and controlled release properties because hydrophilic polymer chains do not

SC

effectively interact with drug molecules [101]. Hence, functional hydrogels were synthesized to deliver ophthalmic drug for oculopathy therapy. For this purpose, functional monomer of

NU

mono-GMA-β-CD and functional crosslinker of MA-β-CD were introduced to hydrogel through copolymerization reaction. The contact angle and equilibrium swelling ratio of hydrogels were affected by MA-β-CD ratio and mono-GMA-β-CD ratio, respectively. All of

MA

hydrogels displayed comparable water loss, appropriate transparency and rheological property of a typical elastomer. Also, the viscoelasticity and surface morphology of hydrogels were

ED

influenced by mono-GMA-β-CD and MA-β-CD ratios. Functional hydrogel comprising β-CD domain revealed superior protein resistance capability and considerably greater equilibrium encapsulated

drug

quantity compared

with traditional hydrogel.

In addition to

the

EP T

performance, the drug release from hydrogel was controlled by means of both mono-GMA-βCD and MA-β-CD ratios. Preliminary in vivo assessment indicated that functional hydrogel contact lens had enhanced influence and effectiveness on dropping intraocular tension relative

AC C

to commercial eye drop. Consequently, it was inferred that functional contact lens was promising for application in oculopathy therapy [101]. The cornea was investigated by slit lamp after it was cultured for 35 days (Fig. 38). Slight edema and some new vessels about marginal corneal were noticed for the cornea treated for 35 days using the drug encapsulated hydrogel contact lens (see Fig. 38a). In untreated cornea, numerous new vessels were homogeneously scattered on the cornea surface, Fig. 8b. The results were also established by HE stain images (Figs. 38c and 38d). The cornea treated using the drug encapsulated hydrogel contact lens for 35 days revealed that evident hyperplasia was mostly observed on marginal corneal as it is shown in Fig. 38c. The untreated cornea exhibited that the hyperplasia was homogeneously scattered over the cornea surface (Fig. 38d). New vessels on the surface of untreated cornea were attributed to intraocular

ACCEPTED MANUSCRIPT hypertension. The slight edema of cornea treated with contact lenses was a problem that was induced through protein deposition and cornea anoxia which might be overcome by improving the usage method and addition of care solution. Because the hydrogel contact lens was fabricated without postprocessing of margin, the irregular margin might have stimulated the tissue which triggered hyperplasia. Thus, the symptom might be relieved by improving the postprocessing method. Consequently, it was concluded that the contact lens had a bright outlook for application in oculopathy therapy [101].

PT

Glaucoma is generally treated by eye drops but this method is not efficient due to fast drug clearance (low residence time) from the ocular surface. On the other hand, contact lenses

RI

are perfectly suitable for controlled drug delivery to cornea; however, it should be taken into

SC

account that introduction of any drug loaded particulate formulation could influence the physical and optical properties of contact lenses [102]. Thus, timolol maleate (TM) loaded

NU

ethyl cellulose nanoparticle-laden ring was implanted in hydrogel contact lenses to allow a controlled drug delivery at therapeutic rates without affecting critical lens characteristics [102]. The TM-implant lenses were achieved through dispersion of TM encapsulated ethyl

MA

cellulose nanoparticles into acrylate hydrogel created as ring or sandwich systems. The TMethyl cellulose nanoparticles were obtained by double emulsion process using diverse ratios of

ED

ethyl cellulose to TM. The XRD patterns showed the TM transformation to the amorphous phase. The in vitro release kinetic data presented a sustained drug release happened within the therapeutic window for 168 h using 150 μg loading. The cytotoxicity and ocular irritation

EP T

assays established the safety of TM-implant contact lenses. The in vivo pharmacokinetic tests in rabbit tear fluid exhibited substantial rise in mean residence time and area under curve using the TM-implant contact lenses compared to eye drop therapy. The in vivo

AC C

pharmacodynamic results in rabbit model proved sustained decrease in intra ocular pressure for 192 h. Accordingly, this investigation validated the auspicious potential of implantation technique to treat glaucoma by means of contact lenses which could be used as a platform for treatment of other ocular diseases. Since the corneal tissue is the most frequently transplanted tissue around the world, recently it was intended to produce a drug-eluting contact lens to be employed as a bandage after keratoprosthesis [103]. Films were prepared by means of poly(vinyl alcohol) (PVA) and CS that were crosslinked by glyoxal (GL) to yield the PVA and PVA-CS films. Also, vancomycin chlorhydrate (VA) drug was incorporated in such systems through soaking. The thermal behavior,

drug release profile,

cytotoxicity, biodegradation, hydrophilicity and

swelling capability of the samples were evaluated. The PVA and PVA-CS films were

ACCEPTED MANUSCRIPT transparent, flexible having smooth surfaces, hydrophilic and could load and release vancomycin for more than 8 h. The biodegradation test in artificial lachrymal fluid using lysozyme at 37 ºC revealed that mass loss was greater for the samples including CS. Furthermore, the samples achieved using CS presented the pore formation which were confirmed by the SEM micrographs. All samples illustrated biocompatible characters when they were in contact with cornea endothelial cells for 24 h. In conclusion, the 70PVA-30CS film could combine the required properties to achieve vancomycin eluting contact lenses in

RI

2.8. Application of chitosan in wound healing

PT

order to avoid inflammation upon corneal substitution.

SC

Wound healing is a complex physiological reaction in a living organism to chemical, physical, mechanical and/or thermal injuries [104]. It is a dynamic procedure where cells and

NU

matrix components act together to facilitate wound regeneration and restore tissue integrity. Wound healing occurs through dynamic and overlapping phases including inflammatory (homeostasis and inflammation), proliferative (granulation, contraction and epithelialization)

MA

and remodeling (maturation) [105]. Nevertheless, when the process does not proceed normally, the healing would not continue afar the inflammatory phase and this is recognized

ED

by the accumulation of great quantities of macrophages and neutrophils along with the exudation of inflammation mediators such as reactive nitrogen species, reactive oxygen species, cytokines and their analogues. Deficiency in wound healing cascade can result from

EP T

critical size skin injury, burn, chemical damage, secondary microbial infections and problems happening due to pathological states such as diabetes [106]. In order to stimulate wound healing, the wound have to be covered using a suitable non-toxic and semi-permeable

AC C

dressing to protect it against external microbial and mechanical stresses. As well, if the wound area maintains a moist environment, the healing process will be initiated [107]. Biopolymers are biocompatible and have the characteristic to swell through liquid uptake by their polymeric networks. Hence, they offer apposite matrixes to stimulate healing cascade with mimicking in milieu moist medium. Among diverse biopolymers, CS is widely used in wound healing as a result of its structural similarity to glycosaminoglycans which is a vital wound repairing macromolecule existing as a component in extracellular matrix (ECM) [108]. Since CS is a biodegradable polymer, its degradation products can initiate synthesis of ECM components signifying its non-toxic nature for in vivo application. Furthermore, it is one of the few polymers showing outstanding antibacterial feature [109]. In addition to a

ACCEPTED MANUSCRIPT simple dressing and film forming ability, it is also employed as complex artificial matrices in tissue engineering. Recently, a water-soluble CS derivative, N-succinyl-chitosan (NSC) was synthesized using succinic anhydride, hydrochloric acid and alkaline CS; then, its capacity to accelerate the wound healing progression was evaluated [110]. The NSC cytotoxicity was examined on L929 cells and its antibacterial activity was assessed through measurement of the inhibition zone. It was found that the NSC solubility was noticeably enhanced relative to CS and NSC

specified

that NSC

PT

was non-toxic having suitable antibacterial potency. The animal wound healing experiment significantly decreased the healing time in comparison to CS.

RI

Histopathological investigation proposed that the principal mechanisms of these phenomena

SC

were associated to NSC capability to stimulate the development of granulation tissue and increasing epithelialization. Overall, it was concluded that the NSC had the potential to be

NU

used in wound healing.

Healing of full-thickness (FT) wounds necessitates further support from native or synthetic matrices in order to assist tissue regeneration [111]. Particularly, a matrix with

MA

optimum hydrophilic-hydrophobic balance is required to experience satisfactory swelling and to diminish bacterial adhesion. In this context, polyurethane diol dispersion (PUD) and the

ED

antibacterial CS were blended in diverse ratios which were self-organized to form macroporous hydrogel scaffolds (MHS) at room temperature upon drying [111]. The AFM and SEM images of MHS indicated the macroporosity on top and cracked surfaces. The FTIR

EP T

spectra displayed that the inter/intra-molecular hydrogen bond interactions formed between the two polymers were responsible for phase separation which was detected in micrographs of blend solutions obtained in the course of the drying process. Also, the influence of phase

AC C

separation on in vitro degradation (hydrolytic, enzymatic and pH dependent) and mechanical characteristics of MHS were examined which proved it would be an appropriate material for wound healing. The in vitro cytocompatibility was established via the proliferation of primary rat fibroblast cells on MHS. Moreover, the MHS was used in in vivo full-thickness wound healing on Wistar rats and the results were compared to the similar Tegaderm™ polyurethane comprising commercial dressing. The MHS-treated wounds revealed faster healing with improved wound contraction, greater collagen synthesis and vascularization in wound area than Tegaderm™. Consequently, it was verified that the MHS was an auspicious sample for application in full-thickness wound healing processes [111]. After 18 days, the subcutaneously implanted C7P3 was retrieved for histological tests (Fig. 39a–c). The H&E staining in Fig. 39d illustrated that the C7P3 was capable to integrate

ACCEPTED MANUSCRIPT with the neighboring tissues by cell infiltration and exhibited the occurrence of matured blood vessels (indicated with an arrow) after 18 days. Any signs of acute inflammations were not seen. Also, toluidine blue stained sections (Fig. 39f) demonstrated small number of mast cells infiltration in C7P3. Such slight inflammatory response was related to the normal wound healing course. The MT staining (Fig. 39e) proved collagen deposition and capsular layer/fibrosis was not observed around the C7P3tissue. C7P3 was also evaluated for FT in vivo wound healing test in a rat model. The C7P3

PT

was adhered to the wound bed because of its inherent great protein adsorption capacity which resulted in biological dressing fixation and avoiding wound exposure to the external medium

RI

(Fig. 39a). Fig. 40a reveals wound healing kinetics and the of wound closure area is observed

SC

in Fig. 40b. The control group displayed 40±1.92%, 65±3.12% and 82±3.91% wound closure on days 7, 14 and 21 but the C7P3 treated groups exhibited improved wound closure values

NU

equal to 55±2.54%, 88±3.41%, and 100±4.12%, respectively. The accelerated wound healing properties of the C7P3 scaffold was corresponded to the exceptional combinatorial characteristics like interconnected porous structure, great protein/water adsorption and pH

MA

sensitive degradation [111].

The influence of policaju (POLI)-CS hydrogel prepared using POLI from cashew tree

ED

(Anacardium occidentale L.) gum and CS was estimated accompanied by low level laser therapy (LLLT) in wound healing [112]. For this purpose, sixty male Wistar rats were allocated to four groups including POLI-CS hydrogel (H), LLLT (L), POLI-CS with LLLT

EP T

(HL) and saline control. The macroscopic assessments were performed by clinical tests and area measurements along with microscopic analysis through histological principles. The H and HL revealed additional esthetical scar tissue and greater wound contraction than control.

AC C

The histopathological analysis illustrated higher existence of fibrin-leukocyte crust in HL and L at day 3, more collagen formation in H, L and HL, low focal necrosis at 7 and 14 days in H, poor neutrophilic exudate in H, L and HL, regression of the vascular neoformation at day 7 in H and identical variation in HL and L. Hence, it was established that POLI-CS caused highly effective healing process and modulated the inflammation and its application along with LLLT potentiated the process. Some chitosan-bentonite nanocomposite (CBN) films were developed using solvent casting process to be employed in wound healing [113]. The physicochemical properties such as folding endurance, thickness, water absorption ability and water vapour transmission rate (WVTR) of the films were examined. The FTIR spectra established the interactions occurred between positively charged CS and negatively charged bentonite. The surface morphology of

ACCEPTED MANUSCRIPT the composite films was observed by SEM images. The WVTR, water absorption ratio, thickness and folding durability of the CBN films were measured equal to 1093±20.5– 1954±51 gm−2 day−1 , 1232±14.58–1688±18.52, 17.50±5–42.50±9.75 μm, and 145.25±2.21– 289.50±0.57 respectively. Because bentonite was very hydrophilic, it highly enhanced the water absorption capabilities of the nanocomposite films. Furthermore, the bentonite existence in the films improved the mechanical strength. Besides, the antibacterial potency of the films was assessed against Gram-positive and Gram-negative microorganisms. All CBN

PT

films exhibited suitable inhibition activity against all the bacteria compared to control. Hence, it was recommended that the CBN films were promising samples as wound dressing

RI

materials.

SC

It was aimed to achieve a composite dressing using collagen, CS and alginate to promote wound healing and avoid seawater immersion [114]. The chitosan-collagen-alginate

NU

(CCA) cushion was obtained by paint coating and freeze-drying which was then attached to polyurethane in order to create CCA composite dressing. Also, the porosity, swelling, degradation and mechanical characteristics of CCA cushion were examined. The effect CCA

model.

The

preliminary

MA

composite dressing on wound healing and seawater prevention was tested using rat wound biosecurity

was

estimated

through

hemocompatibility

and

ED

cytotoxicity experiments. Results proved that CCA cushion had appropriate mechanical and water absorption features. The wound healing ratio was higher in rats treated by the CCA composite dressing than in rats treated using gauze and CS. After five days, the healing rates

EP T

using the CCA composite dressing, gauze and CS were measured to be 48.49±1.07%, 28.02±6.4% and 38.97±8.53%, respectively. The histological images of rats treated by the CCA composite dressing more fibroblast and intact re-epithelialization were perceived and

AC C

the expressions of bFGF, EGF, CD31 and TGF-β were significantly augmented. As well, the CCA composite dressing did not have substantial cytotoxicity but it showed suitable hemocompatibility. Thus, it was found that CCA composite dressing prevented seawater immersion, supported wound healing and it exhibited an appropriate biosecurity. Fig. 41 indicates the wound healing of CCA composite dressing in three treated groups. It was found that after soaking in seawater for 4 h, the dressing covered the wound was yet dry. Thus, the dressing could be utilized for a wounded person working on sea in order to limit the risk of wound seawater immersion syndrome. Also, the wound sites in CCA composite dressing group were healed quicker than both of the gauze and the CS groups (see Fig. 41A). The wounds used the CCA dressing group showed slight inflammation or infection and they appeared as moist and neat. On day 3 after surgery, noticeable differences were not

ACCEPTED MANUSCRIPT observed among three groups because the wounds were moist without any infections. On days 5 and 8, the CCA dressing treated group exhibited more effective wound healing compared to the gauze-treated and the CS-treated groups. From the 11th day, all of the wounds began to contract from the edges and the wound covered using the CCA dressing was contracted more rapidly. Fig. 41B illustrates the wound healing rates indicating on the third day after surgery, the healing rate in CCA treated group was 27.89±6.04% that was greater compared to those of the

PT

gauze negative control (12.46±2.7%) and the CS positive control (23.52±6.13%). On the fifth day, the measured healing rates in CCA treated, CS and gauze groups were 48.49±1.07%,

RI

38.97±8.53%, 28.02±6.4%, respectively confirming the rate in CCA group was very greater

SC

than that of the gauze group. On days 8 and 11, the healing rates in CCA group were 56.69±7.41% and 82.85±7.23%, respectively but those in the CS group were 60.41±3.65%

NU

and 84.11±4.24%, and in gauze group were 43.67±6.05% and 70.78±6.06%. These results exhibit that the healing rates in CCA and CS dressing group were greater than that of gauze group. The healing rates on the 13th day for all three groups were around 90% and there were

MA

not any differences among the three groups. Hence, the CCA composite dressing have improved wound healing efficiency in the early stages of wound healing than CS dressing and

ED

gauze.

The histological data are provided in Fig. 42 showing in day 5 after surgery, the wound areas in CCA treated group contained much more akaryocytes than CS and gauze groups. On

EP T

the eighth day, noticeable granulation tissue growth and partial fibroblasts were seen in the CCA and CS groups. The CCA and CS groups illustrated granulation tissue and the neogenetic epidermis formation in the 11th day. The epidermis and dermis were repaired in

AC C

the all groups in the 13th day and it was looked like normal skin in the CCA treated group. Consequently, the CCA composite dressing revealed high capability for re-epithelialization, well-organized granulation tissue and epidermis/dermis repair [114]. Lupeol entrapped chitosan-gelatin hydrogel (LCGH) films were achieved by means of solution casting technique through blending CS and gelatin using glycerol plasticizer and glutaraldehyde crosslinker [115]. The LCGH films were characterized by SEM, FTIR, differential scanning calorimetry (DSC), equilibrium water content (EWC), WVTR and in vitro release analyses. The SEM images proved the existence of the homogeneous porous structure in both blank and LCGH films. The FTIR and DSC confirmed the presence of lupeol in hydrogels. The LCGH film was smooth, non-brittle and flexible which displayed outstanding swelling capacity. The EWC (85.40%) and WVTR (2228±31.8) were suitable for

ACCEPTED MANUSCRIPT a perfect wound dressing. Also, the biological properties of lupeol were evaluated using antibacterial and antioxidant assays. The antioxidant assay established that lupeol and LCGH film had exceptional antioxidant activities due to scavenging radicals with an enhanced constant rate which was increased with time upon the continuous lupeol release. The disc diffusion method exhibited that the antibacterial activity of lupeol in LCGH film was preserved. The cell viability was estimated by the MTT assay using NIH/3T3 fibroblast cells and it was found that the CGH film obviously had satisfactory non-toxicity and cell viability.

PT

Hence, it was illustrated that CS/gelatin hydrogel film was a perfect delivery system for sustained lupeol release and the LCGH film was an auspicious wound healing material [115].

RI

The RGD peptide sequences can regulate cellular actions through interaction with α5 β1 ,

SC

αv β5 and αv β3 integrin, which influences the wound healing [108]. In a recent work, the RGDC peptide was immobilized onto CS derivative 1,6-diaminohexane-O-carboxymethyl-

NU

N,N,N-trimethyl chitosan (DAH-CMTMC) in order to demonstrate the RGDC-supporting adhesion for improved wound healing [116]. The effectiveness of N-methylation, Ocarboxymethylation and spacer grafting was both qualitatively and quantitatively studied 1

H NMR spectra which showed >0.85 substitution degree for O-

MA

using the FTIR and

carboxymethylation and 0.38 for N-methylation. Also, the glass transition temperatures were

ED

measured for the CS derivatives. The peptide immobilization was done by the sulfhydryl groups by means of sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). The RGDC immobilized peptide onto the DAH-CMTMC was ~15.3 μg/mg for the CS

EP T

derivative that was proved by the amino acid analysis. A substantial increase in human dermal fibroblast viability in vitro during 7 days (viability >140%) proposed that the RGDCfunctionalized CS led to greater wound healing. Besides, proliferation and bio-adhesion

AC C

assays justified that coating of the RGDC-functionalized CS derivatives exhibited in vitro wound healing through increasing fibroblast adhesion and proliferation. The results revealed that the RGDC peptide-functionalized CS offered an ideal substrate for both fibroblast proliferation and adhesion. It was aimed to prepare CS hydrogels containing nerolidol to optimize their antimicrobial and wound healing properties [117]. The hydrogels were achieved by addition of 2 or 4% of the nerolidol to the CS solution. The TGA, DSC and FTIR analysis confirmed the nerolidol incorporation in the hydrogels. Also, direct contact of hydrogels and Staphylococcus aureus bacterium exhibited a synergistic influence in the materials which led to complete bacterial growth inhibition. The hydrogel comprising 2% nerolidol displayed exceptional

healing

activity.

The

emergence

of

re-epithelialization

and

collagen

ACCEPTED MANUSCRIPT reorganization was witnessed on the day 7 of treatment. Hence, the hydrogels ascertained to be favorable as healing and antibacterial substances. In another study, a promising wound dressing was developed using CS cross-linked with genipin by introducing partially oxidized Bletilla striata polysaccharide [118]. The prepared material was coded with CSGB which presented lower gelling time, more homogeneous aperture distribution, greater water retention, required mechanical strength and higher L929 cell proliferation relative to the CS only cross-linked by genipin. Because free

PT

amino groups of CS were partially blocked, the CSGB practically did not indicate antibacterial potency, consequently a bilayer composite of CS-silver nanoparticles (CS-AgG)

RI

was produced on CSGB in order to inhibit microbial growth. The in vivo tests specified that

SC

both CSGB and bilayer wound dressing considerably enhanced the healing rate of cutaneous wounds in mice. The bilayer demonstrated superior mature epidermization with fewer

NU

inflammatory cells on day 7. Accordingly, the bilayer composite was highly promising for wound dressing application.

Recently, CS–hyaluronic acid composite sponge scaffold was developed that was

MA

supplemented with Andrographolide (AND) lipid nanocarriers [119]. One of nanocarriers named NLC4 showed the highest desirability value; hence it was selected as the best

ED

nanocarrier. It had a spherical shape that was 253 nm in diameter, 83.04% entrapment efficacy and a prolonged AND release up to 48h. The NLC4 was incorporated into a CS– hyaluronic acid gel and lyophilized for 24 h to acquire CS–hyaluronic acid/NLC4

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nanocomposite sponge in order to increase AND carriage to wound sites. The sponge morphology was characterized by the SEM micrograph. The nanocomposites exhibited 56.22% porosity and improved swelling character. The in vivo evaluation in rats established

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that the CS–hyaluronic acid/NLC4 sponge boosted the wound healing without scar and amended tissue quality.

It is known that severe oxidative stress in chronic wounds hinders the wound healing. Thus, an antioxidant-loaded hydrogel was fabricated to heal diabetic wounds [17]. For this purpose, composite hydrogels composed of CS, heparin and poly (γ-glutamic acid) in diverse ratios were produced by electrostatic interactions. The hydrogels displayed good 3D network structures and their porosities were diminished as the crosslinking density was improved. The hydrogels indicated suitable swelling capability, usual viscoelastic character and satisfactory mechanical feature in rheological test. The fibroblast proliferation test established that the hydrogels were cytocompatible. Superoxide dismutase was loaded to the hydrogel to achieve a wound dressing with antioxidant activity. It was shown that the dressing applied on diabetic

ACCEPTED MANUSCRIPT rat models accelerated wound healing through stimulating wound closure and collagen deposition. Overall, the hydrogel demonstrated appropriate physical properties and could effectively support repairing chronic trauma in diabetic rats confirming it would be a promising wound dressing. A composite sponge was prepared through physical mixing of hydroxybutyl CS and CS to get a porous spongy material by vacuum freeze-drying method [120]. The macroporous and hydrophilic hydroxybutyl CS composite sponge was produced by the introduction of CS to

PT

hydroxybutyl CS. The composite sponge exhibited greater porosity of ~85%, superior water absorption of near 25 times, improved softness and less blood-clotting index compared to

RI

those of CS and hydroxybutyl CS sponges. The composite sponge with satisfactory

SC

hydrophilic nature absorbed moisture from blood to raise the blood concentration and viscosity, and became a semi-swelling viscous colloid which could block the capillaries. The

NU

cytocompatibility experiments using HUVEC and L929 cells established that composite sponge was not cytotoxic and could stimulate the fibroblasts growth. The composite sponge was made up in order to attain higher antibacterial potency (>99.99% reduction) due to the

MA

hydroxybutyl CS had drawbacks and poor antibacterial activity. Ultimately, the in vivo tests performed on Sprague–Dawley rats illustrated that epithelial cells were attached to the

ED

composite sponge and penetrated to the interior. Furthermore, it was evidenced that the composite sponge had a superior capacity to stimulate wound healing and faster skin glands creation and re-epithelialization.

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Repairing dermal wounds mainly in the diabetic population is an important healthcare problem [121]. The poor wound healing in diabetic wounds can be ascribed to low levels of endogenous growth factors such as vascular endothelial growth factor (VEGF) that generally

AC C

stimulate multiple phases of wound healing. Recently, CS scaffolds were produced by freeze drying and incorporated with plasmid DNA encoding perlecan domain I and VEGF189 and their capacity to support dermal wound healing was investigated in vivo. The plasmid DNA encoding perlecan domain I and VEGF189 loaded scaffolds stimulated dermal wound healing in both normal and diabetic rats. Such treatment enhanced number of blood vessels and subepithelial connective tissue matrix components in the wound beds relative to wounds treated with CS scaffolds including control DNA or wounded controls. Therefore, it was suggested that the CS scaffolds embedded with plasmid DNA encoding VEGF189 and perlecan domain I had the potential to promote angiogenesis and wound healing. It has been known that wound dressings should preserve a moist and alkaline environment to create a protecting barrier against secondary infections and mechanical stress

ACCEPTED MANUSCRIPT and to promote granulation [122]. In an effort, a polymer-based sponge composed of CSsodium hyaluronate-resveratrol (CHR) was synthesized and its regenerative ability was evaluated [122]. The porosity, density and cytotoxicity of the sponges were studied. The in vivo test was carried out on the CHR polymer to decide its potential to stimulate tissue regeneration on a reproducible and measurable skin wound created in an animal model. The skin punch biopsies were harvested from the healed area and were evaluated by the histopathological experiments. Results confirmed that the CHR polymer enhanced the

PT

granulation formation and assisted wound healing along with a bacteriostatic effect. Several flexible and transparent CS-based membranes incorporated with antibacterial

RI

drugs were achieved by a solvent evaporation casting method using a CS floccule suspension

SC

[123]. Glycerin was added as a plasticizer to the CS floccule suspension in order to increase the mechanical properties. The mechanism of membrane creation was attributed to the inter-

NU

and intra-hydrogen bonds formed between CS and glycerol species. Results displayed that glycerol incorporation had an important effect on the characteristics of the CS membranes. Increasing the glycerol amount significantly improved the swelling rate, tensile strength,

MA

wettability and water vapor permeability of membranes. The in vitro enzymatic degradation test exhibited that the CS membrane provided long-term stability irrespective of the glycerol

ED

content. Tetracycline hydrochloride and silver sulfadiazine (AgSD) as the water-soluble and water-insoluble drugs, respectively, were incorporated to the membranes in order to increase antibacterial features. The controlled-release and inhibition zone data pointed out that the

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glycerol reinforced CS membranes loaded by drugs would be promising materials to treat bacterial infections as wound dressings. A wound healing material was produced by means of two marine biomaterials including

AC C

CS and squid ink polysaccharide as carriers and calcium chloride as initiator for coagulation [124]. According to the central composite design using the response surface methodology, the appearance quality and water adsorption ability of composite sponges were used as evaluation indices in order to achieve the optimized preparation conditions and also to estimate the properties of the squid ink polysaccharide-CS (SIP-CS) sponge. The optimized SIP-CS formulation

was

obtained

as

CS

concentration=2.29%,

squid

ink

polysaccharide

concentration=0.55% and calcium chloride concentration=2.82% in a volume ratio of 15:5:2. The SIP-CS was favorable and stuck on the wound, had spongy nature, great tackiness and absorptivity. Rabbit ear arterial, hepatic and femoral artery hemorrhage tests pointed out that compared to CS dressing and absorbable gelatin, shorter hemostatic times and smaller bleeding volume was measured. Besides, the SIP-CS absorbed high quantity of hemocytes

ACCEPTED MANUSCRIPT which led to fast hemostasis. Healing zones and wound pathological pieces in scalded New Zealand rabbits illustrated that the SIP-CS supported wound healing more quickly than CS and superior than commercially accessible burn cream. Amphiphilic CS salt, i.e. CS oleate (CS-OA), was used to physically stabilize lemongrass antimicrobial nanoemulsions (NE) by a mild spontaneous emulsification method [125]. Both oleic acid and CS are known as effective materials in wound healing, thus CS-OA was applied to encapsulate alpha tocopherol (αTph) in NEs that will be used to heal skin

PT

wounds. One developed NE formulation displayed ~220 nm dimensions, αTph concentration of up to 1 mg/ml and 36% drug loading. The two CS-OA and αTph NE supported

RI

proliferation of keratinocytes and fibroblast cells, and the ex vivo skin biopsies proved that the

SC

CS-OA was suitable having antioxidant activity for topical use in wound healing. The αTph stability was more enhanced through encapsulation by NE spray drying as a powder (up to

NU

~90% αTph residual after three months). The spray drying procedure was optimized to increase αTph recovery and powder yield. The powder was simply re-suspended to deliver the NE that could entirely release αTph.

MA

Moist wounds can heal more quickly than dry wounds and hydrogel wound dressings are appropriate materials to heal the moist wounds because they have hyperhydrous structures

ED

[126]. Chitosan is a favored candidate to prepare hydrogel wound dressings due to its exceptional biological characteristics that can promote wound healing. Some physicallycrosslinked CS cryogels were developed only by freeze-thawing a CS-gluconic acid conjugate

EP T

(CG) aqueous solution for application in wound treatment [126]. The CG cryogels were disinfected by immersion in 70% ethanol before their employment onto wounds. The effect of autoclave sterilization (121 ºC, 20 min) on the features of CG cryogel was studied because

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whole sterilization is one of the important necessities for medical devices. It was established that the optimum gluconic acid content in CG (the number of the loaded gluconic acid units per 100 glucosamine units of CS) was 11 for autoclaving. Increasing the CG cryogel crosslinking level upon autoclaving improved the resistance of the gels against the enzymatic degradation.

Additionally,

the autoclaved

CG cryogels preserved promising biological

characteristics of the pre-autoclaved CG cryogels so that they revealed identical hemostatic efficiency in repairing full-thickness skin wounds. Hence, the autoclavable CG cryogels were highly promising as practical wound dressings. Halloysite is a natural nanotubular clay mineral known as halloysite nanotubes, HNTs, which is chemically similar to kaolinite and as a result of its appropriate biocompatibility, it is an interesting nanomaterial for numerous biological applications [136]. It was aimed to

ACCEPTED MANUSCRIPT develop a nanocomposite based on CS oligosaccharides and HNTs to increase healing in the chronic

wounds

treatment

[127].

A

1:0.05

%wt

ratio

HTNs/CS

oligosaccharide

nanocomposite was prepared by mixing the HTNs powder in 1% w/w aqueous CS oligosaccharide

solution

and

spontaneous

ionic

interactions producing a composition

containing 98.6 %w/w HTNs and 1.4 %w/w CS oligosaccharide. Both HTNs and HTNs/CS oligosaccharide nanocomposite displayed satisfactory in vitro biocompatibility to normal human dermal fibroblasts up to 300 μg/ml concentration and improved in vitro fibroblast

PT

motility by stimulating both proliferation and migration. The HTNs/CS oligosaccharide nanocomposite and its two individual components were examined for their healing capability

RI

in a murine (rat) model. The HTNs/CS oligosaccharide led to superior skin reepithelization

SC

and reorganization compared to separate HNTs or CS oligosaccharide. Consequently, the

2.9. Application of chitosan in bioimaging

NU

nanocomposite could be used as a medical agent for wound healing application.

Molecular imaging is a promising non-invasive method to evaluate biochemical and

MA

biological processes occurred in living organisms. Using the X-ray technique in medical imaging, several noninvasive methods have been developed and effectively employed in

ED

various applications such as clinical diagnosis, drug discovery and cellular biology [12]. Hence, these technologies can increase understanding the drug activity and disease in the course of preclinical and clinical drug design/development. Molecular imaging procedures

EP T

have advantage over more conventional readouts like immunohistochemistry which could be described as they can be carried out in the intact organism with enough temporal and spatial resolution to study biological processes in vivo. Besides, molecular imaging leads to a non-

AC C

invasive and repetitive assay of identical living matter by means of the same or different biological imaging tests at diverse time points. Consequently, it exploits the statistical influence of longitudinal assays and decreases the cost and number of necessary animals [128].

Typically, molecular imaging uses particular molecular probes and inherent tissue properties as the basis of image contrast. It has the capability to recognize a combination of data on cell biology, initial detection and characterization of diseases and treatment evaluation. Also, it can help to choose effective drugs that probably appear to be fruitful but to stop using drugs that seem to be failed. Chitosan is a non-antigenic and hydrophilic biopolymer which is non-toxic to mammalian cells; besides, CS composites are biomaterials

ACCEPTED MANUSCRIPT possessing appropriate physicochemical, mechanical and functional properties that can be used in biomedical imaging applications [129]. Recently, a CS-based biosensing material modified with carbon dots (CDs) was prepared for vitamin D2 detection [130]. The CDs were synthesized by microwave pyrolysis method and characterized by TEM images, Raman, FTIR and UV-VIS spectra. Then, the CDs were added to the 1% acetic acid CS solution in order to afford the carbon dots-CS (CD-CS) composite and a thin film of the CD-CS composite was deposited on indium-doped tin oxide

PT

(ITO) glass substrate (CD-CS/ITO) through drop casting process. The composite film surface was studied by AFM, static contact angle and cyclic voltammetry measurements. The CD-

bovine

serum albumin

to

achieve BSA/Ab-VD2 /CD-CS/ITO

bioelectrode.

The

SC

and

RI

CS/ITO surface was additionally modified by immobilizing vitamin D2 antibody (Ab-VD2 ) electrochemical response of the bioelectrode to vitamin D2 antigen (Ag-VD2 ) was performed via differential pulse voltammetry. The biosensing electrode displayed linearity using 10–50

NU

ng.mL−1 of Ag-VD2 . The sensitivity was 0.2 μA.ng−1 mL cm−2 , detection limit was 1.35 ng mL−1 and the shelf-life of biosensor was ~25 days.

MA

It is known that the naturally plentiful CS biopolymer demonstrates pH-sensitive feature within a narrow pH range. Binding hydrophobic moieties to CS chains produces modified CS

ED

polymer with further adjustable pH sensitivity [131]. In this context, near-infrared (NIR) photoluminescent Ag2 S QDs capped by long-chain carboxylic acid were synthesized and conjugated with CS through esterification reaction [131]. The doxorubicin (DOX) anticancer

EP T

drug having affinity for the hydrophobic oleoyl groups was encapsulated into the QDs to achieve Ag2 S(DOX)@CS nanospheres. The in vitro and in vivo tests exhibited that the nanospheres had great antitumor efficiency and released DOX at lower pH in tumor cells. As

AC C

well, the strong NIR signal resulting from the entrapped Ag2 S QDs led to real-time monitoring the distribution of nanospheres in body. To examine the antitumor activity of Ag2 S(DOX)@CS nanospheres, free DOX, Ag2 S@CS nanospheres, Ag2 S(DOX)@CS nanospheres and PBS control were intravenously injected every other day to BALB/c mice having HeLa tumor via tail vein. The body weight and tumor volume of the treated mice were measured in 12 days. Fig. 43a exhibits that the average tumor volume of the Ag2 S@CS treated mice was similar that of the mice treated by PBS at day 12 post-injection. In mice treated using the Ag2 S(DOX)@CS, the tumor growth was highly inhibited during the same period reflected by 48.5% decrease in tumor volume relative to the control. The Ag2 S(DOX)@CS nanospheres were delivered to the tumor place by the influence of enhanced permeability and retention (EPR) and then they were

ACCEPTED MANUSCRIPT internalized the tumor cells. The DOX release was triggered as the nanospheres were entered the cellular compartments including early/late endosomes and lysosomes. The nanospheres sensitivity to the pH variations expedited fast leakage of the nanospheres from the endolysosomal system and decreased the multiple drug resistance (MDR) effect. Fig. 43b indicates that the mice treated differently exhibited only minor body weight changes after 12 days which proved the Ag2 S(DOX)@CS nanospheres could be well-tolerated without having severe side effects.

PT

Distribution of the Ag element in the tumor bearing mice was studied at diverse time intervals after injection of Ag2 S(DOX)@CS nanospheres, Fig. 43c. The Ag amount in the

RI

tumor was very greater than those in five major organs signifying the nanospheres intended to

SC

be accumulated in the tumor and this was related to the EPR influence. The Ag amounts in liver and spleen were greater compared to those of stomach, heart and kidney reflecting rather

NU

higher affinity of the nanospheres for spleen and liver. Furthermore, the Ag amounts in the tumor and the organs started to drop after 6 h upon metabolism. Compared to those in the organs, decreasing the Ag level in the tumor was less noticeable demonstrating extended

MA

retention of the nanospheres in the tumor.

The non-invasive in vivo NIR imaging of nude mice was performed in order to examine

ED

the distribution of Ag2 S(DOX)@CS nanospheres employed for therapeutic purposes in a living body. The nanospheres were dispersed in PBS and administered to anesthetized nude mice through the tail vein injection. At various time intervals post-injection, the mice were

EP T

imaged upon 808 nm excitation. The NIR fluorescence related to the nanospheres in the mice was simply monitored, Fig. 43c. The nanospheres were distributed in the mice bodies by blood circulation after 6 h post-injection, Fig. 43c (i). The fluorescence from the tumor was

AC C

stronger compaed to those of other areas in the mice bodies including spleen, liver and heart. After 12 h post-injection, the fluorescence intensity was yet higher signifying the nanospheres were accumulated in the tumor through the EPR effect (see Fig. 43c(ii)). The fluorescence signals of the mice bodies were yet observed after 24 h along with decreased intensities (Fig. 43c (iii)) which were correlated to the nanospheres clearance through metabolism. Nevertheless, the fluorescence intensity was still high at the tumor site illuminating the nanospheres could remain in the tumor site for an extended time. Additionally, a tumor was harvested from a mouse at 24 h post-injection and imaged, Fig. 43c (iv). Five key organs of this mouse were harvested (see Fig. 43c (v)). The in vivo and ex vivo images established that the Ag2 S(DOX)@CS nanospheres were accumulated in the tumor after injection to a mouse body and released the anticancer drug in intracellular environment to inhibit the tumor [131].

ACCEPTED MANUSCRIPT Highly fluorescent graphene quantum dots (GQDs)-CS (GQDs-CS) hybrid xerogels were simply synthesized and their morphology was controllable through changing the GQDs contents within the xerogels [132]. The GQDs-CS demonstrated a three-dimensional porous network as the GQDs content was 43wt% in the xerogel that was valuable for drug loading and sustained release. It was found that the GQDs-CS could be used for in vivo imaging due to it exhibited strong blue, green and red luminescence by excitation of different wavelengths. Besides, the pH-induced protonation and deprotonation of the –NH2 groups on CS chains

PT

could lead to a pH-dependent drug delivery by the GQDs-CS hybrid xerogel. The imaging flow cytometry as an effective statistical method was used to study the

RI

degradation influence on the biological properties of trimethyl CS (TMC)-based nanoparticles

SC

(NPs) [133]. High transfection efficiency requires an exact balance between NPs stability and degradation. The biodegradation rate of the TMC NPs was changed through variation of the

NU

degree of acetylation (from 4 to 21%) of the polymer producing NPs with diverse enzymatic degradation rates. Although degree of acetylation did not influence the NP size, charge and its capability to protect plasmid DNA, TMC NPs with an intermediate acetylation degree of 16%

MA

presented the highest transfection efficacy. For all of formulations, key steps of the NPmediated gene delivery process were monitored including NP-cell membrane association,

ED

internalization and intracellular transferring such as plasmid DNA transport to the nucleus. The NP cytotoxicity was determined via quantification of cell apoptosis. Hence, it was proved that the biodegradation rate of the NPs affected their intracellular trafficking and accordingly

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their efficacy to transfect cells confirming changing the polymer acetylation degree can modulate the NPs to achieve diverse degradation rates and bioactivities. In addition, this

design.

AC C

approach ascertained to be an appreciated tool in the early phases of nucleic acid vector

3. Pharmaceutical application of chitosan in food industry 3.1. Application of chitosan in food protein binding Food enrichment with proteins and peptides as bioactive compounds has gained great attention but there are some problems in introducing such functions into food, one of which is their instability in the human gastrointestinal tract (GI). Encapsulation technology can be used a valuable method to overcome this challenge [134]. It was recommended that, for systemic absorption into the human GI tract, proteins/peptides must be carried to a lower part of the intestine (ideally the colon) where lower protease activity would expedite preservation of the proteins/peptides and intact bioactivity prior to the systemic absorption. Nevertheless, a large

ACCEPTED MANUSCRIPT peptide difficulty moves through the mucosal barrier to be absorbed intact by the colon. Consequently, it is needed to use membrane disruptors and/or absorption enhancers. In a recent study, the capabilities of encapsulation systems were examined to recognize gastric protection and sustained release in the intestine [134]. For this purpose, bovine serum albumin (BSA), whey protein isolate (WPI), insulin and a casein hydrolysate were encapsulated in CSpolyphosphoric acid (PPA) beads. Then, the in vitro protein release from the beads was assessed in simulated intestinal fluid (SIF, pH=7) and simulated gastric fluid (SGF, pH=3).

PT

Great entrapment efficiencies were attained for intact proteins (>95% in all cases) whereas lower entrapment was measured for the casein hydrolysate (~50%) which were perhaps due to

RI

physical/steric entrapment of the proteins in such CS-PPA beads. Inhibited BSA release was

SC

obtained with low PPA concentration in both SIF and SGF. The WPI and insulin were slowly released in SIF and efficiently preserved in SGF. Peptides from casein hydrolysate were

NU

incompletely (~35%) while rapidly released in SGF with no additional release in SIF. Thus, it was indicated that CS-PPA beads were promising systems for lower gastrointestinal delivery of bioactive proteins.

MA

Ferritins are proteins which can store thousands of iron ions in their internal cavities [135]. Every ferritin contains same or dissimilar twenty-four subunits that are assembled as a

ED

shell-like structure having an inner size of 8 nm and an external diameter of 12 nm. Due to the nano-sized inner cavity as well as the reversible assembly property of the ferritin, small bioactive molecules can be encapsulated in the ferritin cage. Subsequent to the encapsulation,

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food nutrients can be functionalized through the ferritin shell in order to investigate their solubilization, stabilization and targeted delivery characteristics. The inner and outer surfaces of ferritin cage offer twenty interfaces for the encapsulation and transport of food nutrients.

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However, traditional methods for the production of ferritin-nutrients shell-core nanoparticles commonly use acid/alkaline pH transition which possibly leads to the loss of activity for the food nutrients or the creation of insoluble aggregates. Thus, in order to overcome these drawbacks, a facile one-step process was applied to achieve red bean seed ferritin (RBF)EGC-CS (REC) nanoparticle by means of thermal treatment at 55 °C [135]. It was pointed out that the apoRBF was relatively uncoiled with 5.3% decrease in α-helix content which was due to 55 °C treatment. The EGC molecules spontaneously permeated to the ferritin inner cavity with 11.8% (w/w) encapsulation ratio. Also, the thermal treatment assisted the attachment of CS to the outer surface of the ferritin via electrostatic interactions (binding constant=4.7×10 5 M-1 ). The transmission electron microscope and dynamic light scattering data proved that the REC was 12 nm in diameter and 7.3 nm in hydrodynamic radius that was mono-dispersedly

ACCEPTED MANUSCRIPT distributed. As well, the CS decorated onto the apoRBF enhanced the EGC stability through weakening the apoRBF degradation by digestive enzymes in simulated gastrointestinal tract. Hence, the REC shell-core nanoparticle can encapsulate and deliver functional molecules under benign conditions without great pH variations. The complex coacervate formation was examined between CS and canola protein isolate (CPI) extract achieved from canola meal [16]. The yield of CPI-CS complex coacervates were affected by various factors including pH, CPI-to-CS ratio and strength of the

and

un-cross-linked

complex

coacervates

were

PT

electrostatic interaction. Also, the thermal characteristics of the transglutaminase cross-linked investigated.

The

optimum complex

RI

coacervation between CS and CPI was happened in the pH range of 5.8-6.2 using the CPI-to-

SC

CS mass ratio of 16. The highest denaturation temperature and the denaturation enthalpy of CPI in CPI-CS complex were greater than those of the free CPI demonstrating the CPI

NU

thermally stability was increased upon complexation. The thermal stability of the coacervates was more improved once they were cross-linked using the transglutaminase. Higher CPI thermal stability in CPI-CS coacervates specified that CPI-CS coacervates would be

MA

appropriate candidates to encapsulate thermally sensitive pharmaceutics and food ingredients. Thymol nanoemulsions were achieved by spontaneous emulsification, ultrasound and a

ED

combination of both procedures [136]. The most appropriate nanoemulsion in terms of polydispersion and size was spontaneous emulsification where thymol was effectively encapsulated that could inhibit Botrytis cinerea using 110 ppm of thymol. Also, 10% dilution

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of this nanoemulsion in water was employed to obtain quinoa-CS film having a heterogeneous and porous microstructure. The tensile strength of the film was considerably lower whereas its mean elongation at break was comparable to that of the control film. As

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well, the water vapor permeability of the film was analogous to that of the control film. The influence of nanoemulsion-thymol-quinoa protein/CS coating was assessed on mould growth in inoculated cherry tomatoes. The tomatoes having this coating and inoculated with Botrytis cinerea displayed a great decrease in fungal growth after 7 days at 5 °C compared to the control samples (tomatoes without coating and those coated with quinoa protein/CS).

3.2. Application of chitosan in preparation of antimicrobial food additives Food borne diseases are one of important reasons for illness and death worldwide although great progress has been achieved in understanding the infections caused by pathogenic microorganisms, performing more exact control measures and strict controls to produce commercial foods [137]. Moreover, food decomposition as a result of microbial

ACCEPTED MANUSCRIPT action will lead to economic losses. Thus, food preservation is a challenge in food industry which results in using usual food preservatives like benzoates, nitrites and sodium metabisulfite having an extensive history of safe application [137]. Nevertheless, occasional allergic reactions observed in sensitive persons and the possible creation of toxic by-products using several preservatives that may be carcinogenic (such as nitrosamines from nitrites) result in worries among users due to their probable negative health effects. Therefore, there is

preservatives or consuming preservatives with restricted risks. Antimicrobial films and

coatings are

PT

a growing consumer demand to use foods having no or lower concentration chemical

promising materials to control foodborne

RI

pathogens infecting food products [138]. Natural antimicrobial agents are commonly used as

SC

food preservatives in order to increase microbiological quality and safety and to prolong the food shelf life. Numerous natural compounds are applied the food industry as antimicrobials

NU

against pathogenic microorganisms and spoilage [138]. The antimicrobial compounds can be categorized to six groups considering the succeeding criteria including (1) biosynthesis; such compounds are synthesized by ribosomes or they are primary/secondary metabolites; (2)

MA

biological source; created by bacteria, animals (vertebrates and invertebrates) and plants; (3) biological functions like antifungal, antibacterial, antiparasitic and insecticide compounds; (4)

the chemical materials and

ED

molecular features; based on size, charge and hydrophobicity; (5) structure and composition; biomolecules with diverse topologies and (6) molecular

objectives; either intracellular or extracellular.

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Diverse antimicrobial compounds formed by plants and bacteria are employed in food products as biopreservatives since they can prolong the food shelf life. For instance, bacteria can produce antimicrobial materials to inhibit other bacteria, thus they are beneficial to

AC C

control bacterial death and against pathogenic microorganisms in food. Such antimicrobial agents are primarily formed through Gram-positive bacteria such as lactic acid bacteria [139]. Furthermore, natural antimicrobials are of great attention due to they are known to be healthy and safe by customers. Some natural antimicrobials showing suitable antimicrobial activities are essential oils including cinnamon oil, eugenol and thyme oil [140]. Antimicrobial films/coatings loaded by essential oils have frequently been prepared. For instance, coatings fabricated using with 1 g/100 g CS and 3 g/100 g lemon oil considerably decreased the fungal decay of strawberries stored at 5 ºC for 3 days, compared to that of uncoated strawberries [141]. Nisin is the most generally employed bacteriocin in commercial processes. As well, pediocin has been applied in food products. However, several factors unfortunately decrease

ACCEPTED MANUSCRIPT its antimicrobial efficacy in foods [142]. Different factors affect the antimicrobial activity of bacteriocins

including

the

appearance

of

bacteriocin-resistant

bacteria,

conditions

destabilizing the biological effect of proteins like the existence of proteases and oxidation routes, inactivation through other additives, binding to food constituents (for example, protein surfaces and fat particles), low solubility, non-homogenous distribution in the food (if the antimicrobial compound dispersed uneven in the food matrix, it will leave different bare areas that are prone to the microorganism growth) and pH effect on bacteriocin efficacy and

PT

stability.

It is known that combination of lauric arginate (LAE) and cinnamon oil (CO) leads to

RI

synergistic antimicrobial influence on Gram-positive bacteria whereas antagonistic impact on

SC

Gram-negative bacteria [143]. Also, it was found that ethylenediaminetetraacetate (EDTA) could increase the LAE activity and overcome the antagonistic influence of combined LAE-

NU

CO. hence, recently, physical and antimicrobial characteristics of CS films containing LAE, CO and EDTA were investigated [143]. The thickness of CS films was significantly increased by incorporation of antimicrobial materials. The yellow color of films was enhanced but their

MA

water solubility was decreased when the CO concentration was increased. The water vapor permeability of films was greater after incorporating the antimicrobials. Further, the presence

ED

of antimicrobials in CS films dropped the tensile strength while did not affected the elongation amount. The inhibition zones of films loaded by antimicrobials against foodborne pathogens were much greater relative to those of films only containing CS. the EDTA

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addition improved the antimicrobial activities of films including LAE but binding to CO decreased the LAE diffusion from films to inhibit the bacterial growth. Thus, such

safety.

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antimicrobial films containing LAE, CO and EDTA displayed capability to enhance the food

In the recent years, using films and coatings to preserve foods is of great interest due to the development of biopolymeric materials including essential oils as antimicrobial agents in foods in order to substitute synthetic additives [144]. In this context, the influences of composition parameters on antimicrobial potency and characteristics of films fabricated using modified CS comprising diverse types of carvacrol nanoemulsions were investigated [144]. Also, the concentrations of biopolymer and carvacrol nanoemulsion in the film forming dispersions

were

changed

to

increase

the

antimicrobial effect

against

two

model

microorganisms (Listeria innocua and Escherichia coli). The surface hydrophobicity was varied to obtain the optimum conditions for the prepared systems. It was indicated that emulsion formulations had a substantial influence on the inherent antimicrobial activity and

ACCEPTED MANUSCRIPT their interactions with the modified CS matrix could affect properties of films and their bactericidal activities. The two most active emulsions achieved by a combination of glycerol monooleate and polysorbate 20, and whey protein isolates, respectively, added to the modified CS films noticeably enlarged the inhibition zones against L. innocua and E. coli from 7.2-7.4 mm (modified CS alone) to 13.4-16.1 mm, along with preserving the surface hydrophobicity of the films.

Compared

to

the film containing pure carvacrol, the

incorporation of essential oil nanoemulsions into the films created more homogeneous films

PT

having better appearance.

In another work, the CS based films were developed by incorporation of diverse

RI

concentrations of flour plus microparticles of olive pomace flour [10]. The mechanical,

SC

barrier, antioxidant and optical properties of the films were evaluated. Also, the protective influence of films was assessed against nut oxidation. Addition of the olive residue flour into

NU

the CS matrix changed the morphology and produced more rough and heterogeneous films. The incorporation of 10% of olive microparticles highly increased the tensile strength (22.40±0.22 MPa) of films without changing their original features. The flour and the olive

MA

microparticles enhanced the antioxidant activity of the films which was related to the flour concentration or microparticles loaded into the films. The films containing 30% flour or

ED

microparticles were efficient as protective packaging against the nuts oxidation during 31 days. Therefore, the fabricated packages were sustainable considering the materials used for their preparation, their biodegradability and the antioxidants naturally attained from waste.

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Some gelatin-CS films were prepared containing nanoemulsions loaded by several active compounds including N1: canola oil, N2 : α-tocopherol/cinnamaldehyde, N 3 : αtocopherol/garlic oil and N4 : α-tocopherol/cinnamaldehyde and garlic oil [145]. The films

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were fabricated through the casting technique using 5 g N 1,2,3,4 /100 g biopolymers and glycerol as a plasticizer. Then, the physicochemical, antioxidant and antimicrobial properties of the films were assessed. The moisture content (18% w/w) and thermal characteristics of all films were comparable. The water solubility and light transmission at 280 nm of the nanoemulsion containing films were substantially decreased compared to those of the control. The film incorporated with N 1 was the roughest and exhibited the greatest opacity, elongation at break and stiffness decrease, while the films loaded with N 3 and N 4 illustrated the lowest tensile strength and swelling capacity, respectively. Also, films containing nanoencapsulated active agents were highly active against Pseudomonas aeruginosa and displayed great antioxidant activity. Consequently, it was revealed that such nanoemulsion loaded films had the ability to be used as packaging materials to enhance the food shelf life.

ACCEPTED MANUSCRIPT Because nanoencapsulation may enhance the antimicrobial capacity of nisin when used in foods such as lean beef, nisin-loaded nanoparticles were achieved through ionic gelation of alginate and complexation with CS [146]. Also, response surface methodology was applied to find optimized formulation. For the nanoparticles, the z-average was obtained equal to 66.4±8.9 nm, the encapsulation efficiency was 36.1±0.6% and the zeta potential was measured to be -31.7±2.6 mV. The in vitro test on growth inhibition of Listeria monocytogenes at 4 °C exhibited sustained antimicrobial ability of nisin-loaded nanoparticles

PT

for 21 days. Besides, 400 and 800 IU/g encapsulated nisin could inhibit L. monocytogenes growth in refrigerated vacuum-sealed beef samples for 10 and 24 days, respectively, but free

RI

nisin inhibited the microbial growth merely for 4 and 17 days, respectively. Overall,

SC

experimental design was employed to afford nisin-loaded nanoparticles that could be applied in food industry as an antimicrobial agent in order to prolong the shelf life of lean beef.

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As edible films and coatings not only act as barriers of water vapor, gases and volatile compounds but also they can carry functional ingredients, composite edible films were prepared using CS and zein as and supplemented with dicarboxylic acids (succinic acid and

illustrated

MA

adipic acid) and phenolic compounds (gallic acid and ferulic acid) [147]. The composite films superior mechanical and water vapor barrier characteristics. The recovery

ED

percentage of dicarboxylic acids and phenolic compounds from composite films were 48-65% and 71-84%, respectively. As well, the composite films revealed antioxidant activities. The antimicrobial potencies of composite films were proved using Escherichia coli and

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Staphylococcus aureus microorganisms.

It is known that practical application of CS-essential oil blend films has been limited because the extraction of essential oils from plants is not economic [148]. Thus, it was

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attempted to prepare CS films blended with low cost and commercially accessible fats and oils that are consumed in daily human foods such as olive, sunflower and corn oils, butter and animal fats [148]. It was shown that the biological, physicochemical and mechanical features of CS blend films were affected by adding the oils and fats. The CS-olive oil film exhibited superior surface morphology and greater thermal stability compared to the films with other unsaturated oils. The tensile strength, Young’s modulus and elongation at break were enhanced by 57.2, 25.1 and 31.7% for CS-olive oil film, respectively. The highest antibacterial activity was observed for the CS-olive oil blend film that was nearly equal to that of commercial antibiotic gentamicin. Hence, the edible films prepared by incorporation of natural fats and oils in CS could be used as low cost and environmentally friendly packaging materials.

ACCEPTED MANUSCRIPT Nisin-loaded CS-monomethyl fumaric acid (CM-N) nanoparticles were ahieved as a direct food additive [149]. For this purpose, CS was modified by monomethyl fumaric acid (MFA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The CS-loaded nisin (CS-N) and CM-N nanoparticles were formed by ionic interactions occurred between the positively charged amino groups of CS and CS-MFA and negatively charged tripolyphosphate ions. The CS-MFA exhibited 8.38±0.02% substitution of the amino groups. The CS-N and CM-N nanoparticles yields were 81.64 and 76.83% and nisin encapsulation efficiencies were

PT

71.48±0.48 and 60.32±0.63%, respectively. The average particle sizes of CS-N and CM-N nanoparticles were 134.3 and 207.9 nm and the zeta potential values of CS-N and CM-N

RI

nanoparticles were +39.4 and +31.5 mV, respectively. The antibacterial activity of the CM-N

SC

against foodborne pathogens in orange juice after 48 h incubation was significantly greater than those of other samples. Accordingly, the CM-N nanoparticles were suitable materials for

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application in food industry as antimicrobial agents.

In another study, CS-based edible films were fabricated through supplementation of Berberis crataegina DC.'s seed oil and fruit extract to the CS matrix [150]. The films were

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characterized by both physiochemical (DSC, SEM, UV–vis, FT-IR, contact angle and mechanical test) and biological (anti-quorum sensing, antioxidant and antimicrobial) methods.

ED

The CS-fruit extract film indicated greater thermal stability, antimicrobial, antioxidant and anti-quorum sensing potencies than other films. Also, incorporation of B. crataegina's seed oil and fruit extract to the CS film outstandingly diminished the UV–vis transmittance whereas

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improved the tensile strengths. The hydrophobicity of the CS-seed oil film was higher (92.64±4.17) than the CS control film (84.67±1.50) but CS-fruit extract film displayed somewhat lower hydrophobicity (73.82±7.42) compared to the CS film. The total

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extraordinary thermal stability, antimicrobial and antioxidant capability of the CS-fruit extract film showed that B. crataegina's fruit extract could be employed as an efficient ingredient to produce edible films with improved physicochemical and biological features. The

antibacterial

characteristics

of

emulsion-encapsulated

and

unencapsulated

isoeugenol were investigated against biofilms of S. aureus, Lis. monocytogenes, Leu. mesenteroides and P. fluorescens in tryptic soy broth and carrot juice [151]. The emulsion encapsulation improved the antimicrobial capacity of isoeugenol against biofilms in media but did not act in carrot juice. Some isoeugenol emulsions coated by CS disrupted the structures of biofilms. Moreover, addition of the surfactant Tween 80 (which is frequently used for the dispersion of oils in foods) decreased the antibacterial activity of isoeugenol. Thus, it is found that common food additives such as surfactants may show an opposite

ACCEPTED MANUSCRIPT influence on the antibacterial action of preservatives. Isoeugenol proved to be a promising agent as a food preservative due to it acted nearly well against planktonic bacteria and biofilms. Antimicrobial edible films and coatings were fabricated using micro-emulsions to diminish populations of foodborne pathogens in foods [152]. Corn-bio-fiber gum (C-BFG) was applied as an emulsifier along with CS and allyl isothiocyanate (AIT) and lauric arginate ester (LAE) were acted as antimicrobial agents. Micro-emulsions were prepared using a

PT

solution composed of 1% CS, 0.5% C-BFG and 1–4% AIT or LAE. The coatings and films created by means of the micro-emulsions exhibited micro-pore sizes ranged from 100 to 300

RI

nm and micro-channels that efficiently trapped the antimicrobials and facilitated their release

SC

from the center to the surface of the films/coatings to enhance their antimicrobial effectiveness. The coatings and films containing 1% AIT dropped population of Listeria

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innocua by more than 5, 2 and 3 log CFU in culture medium, ready-to-eat meat and strawberry, respectively. The coatings and films loaded by 1% LAE decreased the populations of Escherichia coli O157:H7 and Salmonella spp. to above 5 and 2 log CFU in Tryptic soy

MA

broth and strawberry, respectively. Hence, effective antimicrobial materials were developed to lessen food borne pathogens on ready-to-eat meat, strawberries or other foods.

ED

The influences of guarana, cinnamon, rosemary and boldo-do-chile ethanolic extracts and diverse gelatin:CS (GEL:CS) ratios on the microstructural, optical, barrier, mechanical, antioxidant and antimicrobial features of the films were investigated [153]. The two gelatin

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and CS polymers were homogeneously blended in the film matrixes. Increasing the CS proportion enhanced the elasticity of the films and diminished the water vapor permeability which was not considerably decreased by adding the extracts. The blend films exhibited

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suitable antioxidant activities and exceptional growth inhibitions both against Staphylococcus aureus and Escherichia coli bacteria confirming such films could be alternative active packaging materials in the food industry. The antimicrobial activities of pure polymeric films (CS100 and GEL100) and GEL50:CS50 blended films containing diverse extracts against E. coli and S. aureus are presented in Fig. 44. It is seen that the chloramphenicol as antibiotic control has the utmost inhibition activity among other films. The inhibition zone diameter for pure CS films (CS100) revealed growth inhibitions against both E. coli and S. aureus bacteria but the pure GEL films did not exhibit any antimicrobial activities [153]. Antimicrobial biodegradable films were fabricated using thermoplastic corn starch and CS oligomers to develop a package prototype for perishable food products [154]. The

ACCEPTED MANUSCRIPT diffusion assays established that the CS oligomers could migrate from the active film to the aqueous simulant media. Furthermore, oligomers could diffuse from the matrix irrespective of the aqueous medium acidity. The experimental of diffusion assay data were fitted to a mathematical model by estimation of diffusion coefficients at three pH values equal to 3, 5, and 7. The active film was applied to make sachets to package perishable foods like ricotta, strawberries, and flavored breads, which were stored under controlled conditions for 7 days. Antimicrobial capability of active sachets was verified by yeast and molds counting in the

materials could

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stored foods. It was illustrated that incorporation of CS oligomers into the packaging more effectively inhibit microbial growth compared to the spraying

SC

RI

procedure.

3.3. Application of chitosan in preparation of antibacterial food packaging materials

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The ever increasing population has led to growth the food demand, thus it is required to extend active packaging technology in order to enhance the safety, quality shelf-life and of foods [155]. The active packaging materials not only act as inert barriers to external

oxygen,

ethylene,

carbon

dioxide,

MA

conditions but also they can decrease food waste through scavenging/absorbing (moisture, odors,

UV

light

and

flavors),

emitting/releasing

ED

(antioxidants, ethanol, carbon dioxide, sulphur dioxide, preservatives, pesticides, flavors), removing (removal of food component such as cholesterol and lactose) and antimicrobial characteristics as well as controlling the food quality and temperature [156] in order to

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efficiently preserve food.

Nowadays, development of active food packaging using bio-based functional packaging materials incorporated with natural ingredients and active compounds has received great

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interest [157]. It is notable that the growing obligation for sustainability has resulted in the progress in preparation of biodegradable packages using biopolymers containing bioactive compounds acquired from waste materials to give additional value to such products. The biopolymers have numerous benefits compared to polyethylenes such as nontoxicity, fast biodegradability, biocompatibility with other biopolymers, simple interaction with food, capability to serve as vehicles for antioxidants and antimicrobials. Among several natural polymers employed as biodegradable packages, CS is a functional prominent biopolymer due to its exceptional film forming capacity, great mechanical strength, appropriate barrier property along with intrinsic antioxidant and antimicrobial features [158]. In fact, the biobased packaging materials showing antimicrobial and antioxidant features have become common because microbial and oxidation pollutions are foremost difficulties decreasing food

ACCEPTED MANUSCRIPT safety and quality. Moreover, the plant polyphenols can be used as substitutes to synthetic antioxidant and antimicrobial agents. In order to fabricate environmentally benign packaging materials, portable CS-ZnO nanocomposite pouches were achieved by a simple process through changing the ZnO amount [159]. The films were much superior compared to bare CS and some conventional films. Two bacteria generally contaminated

the packed

meat were nominated to illuminate the

antimicrobial effects of the CS-ZnO films. It was found that the antimicrobial efficacy was

PT

linearly associated to the quantity of ZnO nanoparticles added to the composite. The optimum film displayed outstanding antimicrobial potency; thus it was fabricated as a packaging pouch

RI

for raw meat. The pouch presented substantial activity against the microbes in raw meat due

SC

to total microbial growth inhibition on the sixth day of storage at 4ºC. Hence, the pouch could be used as a top-notch material compared to polyethylene bag applied to extend the raw meat

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shelf life.

The capability of the optimized C-2 film was evaluated as a packaging material to preserve raw meat. Before the test, the C-2 films were preserved in air tight polyethylene bags

MA

during 6 months. The C-2 films were prepared as flexible pocket resembling bags by means of cotton yarn using a home-made weaving device (Fig. 45). The shelf life efficacy of the

ED

composite bag was compared to that of synthetic plastic bag (low density polythene) popularly utilized to pack, store and transport meat and other foods in the marketplace. The composite and plastic bags were sterilized through autoclaving at 121 C for 15 min. Then,

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equal quantity of meat was placed into the bags so that each set of bags contained three duplicates of C-2 bag (i.e. A1 SET) and polythene bag (B1 SET) and incubated for 4 days at 4 C. After four days incubation, aerobic plate count was done. This procedure was repeated for

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A2 and B2 sets incubated for 5 days and followed for A3 and B3 sets incubated for 6 days (see Fig. 46) [159].

Recently, CS films incorporated with 0, 2.5, 5, 10 and 20% w/w propolis extract (high in polyphenols) were fabricated [160]. The elongation at break, tensile strength, total phenolic content and antioxidant capacity of films were increased but their water vapor permeability and oxygen permeability were decreased by increasing the propolis amount. Also, increasing propolis extract concentration endowed with a deeper orange color to the films compared to light yellow color of the control film. The capability of the films to inhibit Salmonella Enteritidis, Staphylococcus aureus, and Pseudomonas aeruginosa Escherichia coli was assessed using agar diffusion method. The CS films containing propolis extract inhibited all of bacteria beneath the films. The FTIR spectra of the films were changed upon adding

ACCEPTED MANUSCRIPT propolis extract indicating some interactions were happened between CS and propolis polyphenols. The appropriate oxygen and moisture barrier, mechanical properties as well as the antimicrobial and antioxidant activities of the films proved adding propolis extract to the CS films was beneficial and the films could be served as active food packaging materials. In another study, flexible, homogeneous and transparent films were prepared using CS and ellagic acid [161]. For this purpose, different ratios of ellagic acid (0.5, 1.0, 2.5 and 5.0% w/w) were used relative to CS to estimate their UV-blocking features. Additionally, the

PT

photochemical stability of the films was investigated through artificial solar light radiation. These films displayed UVA- and UVB-barrier ability, high mechanical characteristics

RI

(Young’s modulus=3.21-3.57 GPa), moderate water vapor permeability (WVP=2.82-3.70 g

SC

mm m-2 day-1 kPa-1 ), thermal stability up to 215-220 ºC and the highest antioxidant capacity of about 28% by scavenging 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH). All of CS-ellagic

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acid films exhibited antimicrobial activity against food-borne pathogens including Gramnegative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. Some blended films were achieved using poly(vinyl alcohol) (PVA) containing CS by

MA

solution casting and electrospraying techniques [162]. The influence of CS amount was evaluated on the mechanical, thermal, water vapor permeation, oxygen permeability and

ED

antibacterial (against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli strains). Relative to the pure PVA film, the PVA-CS films displayed superior elongation at break, less oxygen permeability, improved water barrier properties and higher antibacterial

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activity, particularly when the PVA:CS weight ratio was 75:25. An active film was developed using CS and kombucha tea (KT) by the solvent casting method and the influence of KT encapsulation (1–3% w/w) was studied on the functional and

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physical features of CS film [163]. The antimicrobial ability of the CS-KT film was assessed against Escherichia coli and Staphylococcus aureus bacteria by agar diffusion technique and its antioxidant activity was evaluated by the DPPH scavenging test. It was found that the KT addition to the CS films enhanced the water vapor permeability from 256.7 to 132.1 g cm−2 h−1 KPa−1 mm and improved the antioxidant activity up to 59%. Additionally, the KT incorporation to the CS film augmented the protective influence of the film against ultraviolet irradiation. The FTIR spectra exhibited that chemical interactions were occurred between CS chains and the polyphenols of KT. The CS-KT film successfully acted as an active packaging and prolonged the shelf life of the minced beef which was established by the inhibition of lipid oxidation and microbial growth (from 5.36 to 2.11 log CFU.g−1 ) during four days of storage. The CS-KT film not only preserved the quality of the minced beef but also

ACCEPTED MANUSCRIPT considerably retarded microbial growth and extended the shelf life of the minced beef meat up to three days. Several CS films containing thinned young apple polyphenols (YAP) were prepared and their mechanical, physical and bioactivity characteristics of were investigated [164]. It was indicated that adding YAP significantly increased the density, thickness, opacity, solubility and swelling degree of CS films but the water vapor permeability, water content and mechanical characteristics of the film were

diminished.

Also,

the antimicrobial and

PT

antioxidant features of the CS films were noticeably improved through YAP addition. The FTIR and NMR spectra confirmed that the interactions occurred between CS chains and YAP

RI

probably had a non-covalent nature. The thermal stability of the films was declined when

SC

YAP was introduced. The XRD patterns of the YAP-CS films revealed that the crystalline degree was changed kept pace with that of their thermal stability.

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A CS-TiO2 composite film was produced by incorporating TiO2 nano-powder in CS which exhibited high microbicidal activity against food-borne pathogenic microbes and anticipated to be an auspicious food packaging compound [165]. The SEM image of the

MA

composite film displayed that the TiO 2 nano-powder was uniformly dispersed in the CS matrix. TiO 2 incorporation improved the hydrophilicity, mechanical properties but diminished

ED

the visible light transmittance. The CS-TiO 2 film illustrated high antimicrobial capacity against four strains including Staphylococcus aureus, Escherichia coli, Aspergillus niger and Candida albicans bacteria after incubation for 12 h. Thus, it was provoked that cellular

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substances were leaked through the damaged bacterial membranes which was led to their death. Moreover, the CS-TiO2 film was used in packaging red grapes to avoid microbial infection and prolong their shelf life.

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In another work, cellulose nanofiber (CNF) was modified by rosin and employed to reinforce the polylactic acid (PLA) matrix and the film was coated by CS to obtain a twolayer composite film to be used in antimicrobial food packaging [166]. The FT-IR spectra of rosin modified CNF (R-CNF) exhibited a peak at 1730 cm−1 confirming the esterification of CNF by rosin. The dispersion of R-CNF in the PLA matrix was more appropriate than CNF and the R-CNF loading significantly affected the mechanical properties of the film. It was observed that a percolation network was created using 8% R-CNF loading where the film demonstrated the optimum mechanical features. The antimicrobial assay revealed that the RCNF/PLA/CS film had exceptional antimicrobial capacity against B. subtilis and E. coli microorganisms that was recognized as the synergistic antimicrobial effects of rosin and CS.

ACCEPTED MANUSCRIPT Antimicrobial performance of low/medium-molecular weight CS and organic acids including sorbic acid and benzoic acid and commercially accessible nano-sized sorbic- and benzoic- acid solubilisate equivalents was evaluated and compared with those of commercial mixtures of organic acids applied as meat coatings (Articoat DLP-02® and Sulac-01®) [167]. The antimicrobial tests exhibited that both low and medium molecular weight CS displayed the highest antimicrobial capacity against all tested bacteria with mean minimum inhibitory concentration values of 0.010 and 0.015% w/v, respectively. Hence, it was found that the CS

PT

molecular weight affected its antimicrobial activity. Moreover, the nano-sized solubilisates of sorbic acid and benzoic acid revealed considerably greater antimicrobial activities compared

RI

to their non-nano equivalents.

SC

The influence of CS concentration on the characteristics of biocomposites incorporated with plasticized poly(lactic acid) (PLA) with tributyl o-acetyl citrate was investigated [168]. It

sheets/films

acquired

from

plasticized

NU

was indicated that the tributyl o-acetyl citrate decreased the PLA brittleness and the PLA-CS

biocomposites

exhibited

acceptable

mechanical, transparence and thermal features. The PLA-CS biocomposites were non-toxic

MA

for growth of radish and cucumber seeds. The migration tests were accomplished on two food simulants and one method was modified to avoid using high temperatures near the glass

ED

transition of PLA. It was found that the biocomposites were appropriate materials for the packaging of non-fatty foods with pH> 4.5 at refrigeration temperature. All of the PLA-based materials

presented

satisfactory

antifungal capacity

against

Penicillium

corylophilum

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CBMF1, Aspergillus brasiliensis ATCC 16404 and Fusarium graminearum G87 at the contact surface which was increased by the CS addition. It was observed that only biocomposites comprising CS displayed significant decrease in E. coli and S. aureus

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microorganisms.

In another work, antimicrobial characteristics of CS and CS-ZnO nanocomposite coatings were examined on polyethylene films [169]. Oxygen plasma pretreatment of polyethylene films improved adhesion of 2% CS and nanocomposite coating solutions onto the packaging films. The SEM micrographs indicated uniform coatings were formed on the polyethylene surfaces. Addition of ZnO nanoparticles to the CS matrix caused 42% solubility increase, the swelling was decreased by 80% but the water contact angle was enhanced from 60 to 95° relative to the CS coating. As well, the polyethylene coated by CS-ZnO nanocomposite films totally deactivated and inhibited the growth of food pathogens whereas the CS-coated films only exhibited 10-fold decrease in the viable cells of Escherichia coli, Salmonella enterica and Staphylococcus aureus after 24 h incubation relative to the control.

ACCEPTED MANUSCRIPT The propolis and CS were combined to achieve a completely bio-based active food packaging material [170]. To do this, propolis glycolic extract was added as antioxidant and antimicrobial agent having polyphenols. Two CS polymers with diverse molecular weights were employed as antimicrobial, wet strength additive substitute and polyphenols carrier. The paper was produced using carboxymethyl-/microfibrillated-cellulose at two pH values to examine the polyphenols preservation and paper strength. It was shown that using CS instead of the most frequently used wet strength resin (polyamine polyamide epichlorohydrin)

PT

improved the polyphenols retention more than 10 times. The paper sheets obtained at pH=7 using the highest molecular weight CS by microfibrillated cellulose incorporation exhibited

RI

the highest wet resistance (13.3±1.2%) and wet strength (7.4±0.5 Nm/g). The antimicrobial

SC

activity of the paper was established on thinly sliced raw veal meat and a decrease was

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realized for the inoculated L. innocua after 48 h at 4 °C.

4. Application of chitosan in textiles industry (preparation of antibacterial textiles) Currently, there is an increasing requirement to provide healthy, safe and comfortable

MA

living environment and protection from the infection by pathogenic microorganisms [171]. Therefore, the demand for healthcare/medical textiles and antimicrobial products is ever-

ED

increasing. Thus far, numerous chemicals have been used to endow with antibacterial capacity to textiles including organometallics, inorganic salts, iodo-phors (materials that gradually release iodine), onium salts, phenols and thiophenols, heterocyclics with anionic groups,

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antibiotics, ureas and related compounds, nitro compounds, amines and formaldehyde analogues [172]. Nevertheless, some of such chemicals are toxic to humans and cannot be simply degraded in the nature.

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The textile industry seeks environmental processes to be accomplished without using toxic textile chemicals. Since CS is an exceptional material for an eco-friendly textile industry, it is employed in health-care products and in hygiene fabric engineering such as bandages, artificial implants and medical sutures [173]. As well, CS has the capacity to be utilized in the textile field as a dye fixing agent, thickener and binder for pigment printing of cellulosic fabric and to improve the fastness characteristics of dyed textiles [173]. It was reported that the properties of cotton fabric were enhanced by nanochitosan incorporation and consuming natural bioactive compounds including CS as antimicrobial and ecological finishing of textiles. The CS nanoparticles were prepared by ionic gelation method through the interaction of CS and sodium tripolyphosphate in acidic medium [18]. The CS NPs were characterized for

ACCEPTED MANUSCRIPT their size, morphology and zeta potential and then they were applied on cotton fabric by the pad-dry-cure procedure. The nanofinish process was optimized by Taguchi approach and the responses were bending length, recovery angle, fabric tensile strength, absorption time and antibacterial performance against some bacteria. The smallest average particle size was 115 nm and highest zeta potential was +31.3 mV using 0.2%w/v of CS. The SEM image equipped with energy dispersive X-ray (EDX) established the existence of CS NPs on the treated fabric. The treated fabrics displayed durable and satisfactory antibacterial capacities with suitable

PT

textile features.

A series of CS based water dispersible polyurethanes (WDPUs) were synthesized in

RI

three steps [174]. In first step, the NCO end capped PU prepolymer was obtained from the

SC

reaction of dimethylolpropionic acid, polyethylene glycol and isophorone diisocyanate. In second step, the neutralization was performed using triethylamine to achieve neutralized NCO

NU

terminated PU-prepolymer and in the last step, chain extension was accomplished via CS addition to make the dispersion formation by adding calculated quantity of water. The structures of CS-WDPUs were established by the FTIR spectra. The antimicrobial capacities

MA

of the bare weave poly-cotton printed and dyed textile swatches were assessed after CSWDPUs application. It was found that the CS incorporation into the PU backbone

ED

evidently improved the antibacterial capability of WDPUs. Hence, such synthesized CSWDPUs were produced as sustainable antimicrobial finishes using CS as a natural bioactive agent to be used on polyester/cotton textiles.

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An antibacterial coating was developed for cotton fabrics using core-shell particles composed of poly(n-butyl acrylate) (PBA) cores and CS shells [175]. The spherical particles were achieved by a surfactant-free emulsion copolymerization of n-butyl acrylate in aqueous

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CS solution prompted in presence of a little quantity of tert-butyl hydroperoxide. The PBACS core-shell particles exhibited a narrow particle size distribution, an average particle diameter of ~300 nm and very positive surface charges. The TEM images obviously showed the well-defined core-shell morphology for the particles so that the PBA cores were coated by the CS shells. Each particle contained both the PBA homopolymer and the CS-g-PBA copolymer that were characterized by the 1 H NMR and FTIR spectra. The cotton fabric was coated by the PBA-CS particles using a common pad-dry-cure process and its antibacterial efficacy was quantitatively assessed against Staphylococcus aureus by the shake flask technique. The cotton treated with PBA-CS particles illustrated an outstanding antibacterial capacity and bacterial reduction was greater than 99%.

ACCEPTED MANUSCRIPT Chitosan can be used as a safe antibacterial material on textiles however there is a limitation for its durability. Thus, CS was extracted from shrimp shells and employed as antibacterial exhaust finishing agent on grafted bamboo rayon [176]. Then, the antibacterial activity of CS bound bamboo rayon was estimated against Gram negative and Gram positive bacteria. The product exhibited antibacterial capacity against both types of microorganisms which was durable until 30 washes. In another study, CS extracted from waste shrimp shells was applied in finishing

PT

formulation for cotton fabric, accompanied by dimethylol dihydroxy ethylene urea and other chemicals to impart multiple properties like wrinkle free, flame retardant and antibacterial

RI

features [177]. The finished fabrics were examined for textile properties such as bending

SC

length, tensile strength, yellowness index and functional characteristics including antibacterial capacity, crease recovery angle, flame retardancy and ecological properties like formaldehyde

NU

release. The finished fabric exhibited exceptional crease recovery, antibacterial activity and flame retardancy which were preserved to an adequate level even after 20 washes. In addition to the formaldehyde scavenging property, CS obviously endowed with multifunctional

MA

properties to the cotton fabric.

Some polyurethane (PU) dispersions were synthesized by a two-step polymerization

ED

reaction [178]. To do this, a PU prepolymer with NCO termini was achieved using isophorone diisocyanate, poly caprolactone diols and dimthylolpropanoicacid. The PU prepolymer chain was extended using various mole ratios of low molecular weight CS and the aqueous

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emulsion was obtained through addition of appropriate water amount. The structures of CS based PU dispersions were established by the FTIR spectra. The aqueous CS-PU emulsions were applied on the diverse quality bare weave poly-cotton dyed and printed fabric pieces by

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means of pad-dry-cure technique. The physical characteristics of the treated and untreated fabric samples including pilling resistance, stiffness, air permeability, crease recovery angle, tear and tensile strength were evaluated. It was found that the CS incorporation had a prominent influence on the properties of treated fabrics. In another research, environmental biosynthesis of chitosan–neem seed (CS-NS) composite was accomplished through co-precipitation technique using aqueous neem seed extract [179]. Cotton fabrics were treated using citric acid and glutaraldehyde as two diverse crosslinking materials and the synthesized composite was then coated on cotton fabric via chemical binding between the cellulose structure and the composite coating. The antibacterial performance of the CS-NS composite coated cotton fabric and CS-NS composite coated cotton fabric along with crosslinking agents were assessed against the Gram negative and

ACCEPTED MANUSCRIPT Gram-positive microorganisms using agar well diffusion procedure. It was confirmed that CSNS composite with crosslinked coated cotton fabric had greater antibacterial potency than cotton fabric without crosslinking. Thus, the CS-NS composite could be used to prepare medical textiles. Fig. 47(a–e) exhibits the morphologies of untreated cotton fabric, CS-NS composite, CS-NS composite coated cotton, CS-NS composite coated cotton with glutaraldehyde and CS-NS composite coated cotton with citric acid. The CS-NS composite in Fig. 47(a) displays

PT

a multilayered, rough and non-even surface. The untreated cotton demonstrates that there is no deposition of composites, Fig. 47(b). The CS-NS composite coated cotton shows a layer

RI

resembling structure and several places demonstrate the composite deposition in Fig. 47(c).

SC

The CS-NS composite coated cotton with glutaraldehye, Fig. 47(d), indicates a rough surface and good composite deposition over the cotton surface because of using crosslinker. Besides,

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Fig. 47(d) illustrates extra roughness than that of the composite coated cotton along with numerous spherical particles dispersed over the fabric surface. The CS-NS composite coated

fabric upon using citric acid [179].

MA

cotton with citric acid in Fig. 47(e) reveals that the composite is well bound to the cotton

Durable cotton/polyester blended fabrics with multifunctional features were produced

ED

by introducing CS and several metal oxide nanoparticles (MONPs), i.e., TiO 2 , ZnO and SiO 2 to the fabric surface by means of citric acid/sodium hypophosphite as ester–crosslinking and generating anchoring/binding sites (COOH groups) on the ester-crosslinked fabrics surfaces

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[180]. The surface morphology and the existence of MONPs and CS active ingredients on the coated fabrics were confirmed by the SEM images and EDX analysis. The effect of different finishing

formulations

on functional and

performance properties

including antibacterial

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activity, wettability, self-cleaning, UV-protection, resiliency and durability to wash were examined. It was found that the improvement degree of the imparted functional properties was controlled by the loaded composite type and changed in the order of CS-TiO 2 NPs> CSZnONPs>SiO 2 NPs>CS

alone,

plus

substrate

kind

as

cotton/polyester

(65/35)>cotton/polyester (50/50). Furthermore, the durability of the CS-TiO 2 NPs loaded substrates was slightly diminished after 15 washing cycles demonstrating the durable fixation of the composite components on the ester-crosslinked substrates. Hence, such bioactive multifunctional textiles could be employed to produce ecological protective textiles. To prepare durable antimicrobial cotton fabric, carboxymethyl CS was covalently bound to cotton fibers through esterification using the cellulose hydroxyl groups and the silver nanoparticles were attached to the fiber surface via the coordination bonds with the

ACCEPTED MANUSCRIPT amino groups of carboxymethyl CS [181]. The finished cotton fabrics revealed exceptional laundering durability and outstanding antibacterial activities. After 50 sequential laundering, the modified cotton fabrics exhibited acceptable bacterial decrease rates against both E. coli and S. aureus, which were greater than 94%. Therefore, these antimicrobial cotton textiles had lessened safety risk and decreased environmental influence aroused from the silver nanoparticles. The environmental synthesis of CS-based nanocomposites was carried out by

PT

interactions of AgNPs and clay with CS to yield CS-AgNPs and CS-AgNPs-clay nanocomposites which were applied on cotton fabric to endow with them fabulous properties

RI

[182]. The CS-AgNPs-clay nanocomposites explicitly proved that their usage in one–step

SC

treatment process on cotton fabrics caused noticeable appropriate properties such as uniform morphology, great strength, improved thermal stability, effective composite deposition on the

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surface of cotton fabrics, high water absorption, flame retardancy, antimicrobial capacity, UV protection and controlled release of fragrance. The treatment of cotton fabrics using these nanocomposites was stable against washing after 20 washing cycles. Consequently, the

MA

environmentally benign synthesis of CS-AgNPs-clay nanocomposites was a promising method to achieve multifunctional finishing textiles.

ED

Cotton fabrics treated by the CS/AgNPs and CS/AgNPs/clay nanocomposites were compared to the cotton fabric only treated with CS indicating the color varies from colorless to yellow and yellow darkness, respectively. The surface morphologies of the untreated and

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treated cotton fabric using CS, CS/AgNPs and CS/AgNPs/clay studied by FE-SEM images are exhibited in Fig. 48 confirming the untreated sample shows a rather smooth surface, Fig. 48(A, a) indicating there are not any nanoparticles on the surface. Treatment with CS solution

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causes the CS film to cover the fabric surface (alongside the formation of CS film) and the surface to be rough by the influence of CS polymer, see Fig. 48(B,b). The SEM micrographs of cotton fabrics treated by CS/AgNPs nanocomposite are provided in Fig. 48C. It is apparent that the AgNPs have been deposited on the cotton fabrics surface. Furthermore, the SEM image at higher magnification (8000X) points out that the AgNPs are evenly dispersed on the fabrics. The CS/AgNPs nanocomposites are spherical having small diameters and the cotton fabric treated by colloidal dispersion of hybrid CS/AgNPs/clay composite is covered by aggregated nanocomposite and a rough surface is achieved. The SEM images (Figs. 48D and 48d) exhibit the creation of aggregated clay minerals over the cotton fabrics surface [182]. A facile process was established to prepare the super-hydrophobic antibacterial textile for biomedical purposes [183]. For the coating formulation, the spraying of nanoparticles

ACCEPTED MANUSCRIPT dispersion on the textile was done to obtain a multiscale textured layer on top of cotton fabric. The antibacterial activity of coating was confirmed using CS-based nanoparticles. The nanoparticles were fabricated by electrostatic interactions of amino groups of CS and negatively charged fluoro anions. The relative number of fluoro anions per CS unit had a crucial role on the aggregates structures in the coating, the wettability and durability of coatings in contact with aqueous environment. The surface of wool fabric was modified using anhydrides in order to graft the CS

PT

polymer [184]. The weight gain, antifelting and antibacterial characteristics of the CS graftedacylated wool fabric were evaluated. The wool fabrics were acylated by two anhydrides,

and

dimethylsulfoxide.

The

influences

of solvents,

anhydrides,

anhydride

SC

formamide

RI

succinic anhydride and phthalic anhydride in two diverse solvents including N,N-dimethyl

concentration, reaction time and liquor ratio on wool acylation were explored. The CS was

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grafted to the acylated wool and the influences of CS concentration, pH and reaction time on grafting CS to the acylated wool were assessed. The FTIR, DSC, SEM and weight gain analyses proved that CS was grafted to the acylated wool by the covalent bonds. The grafted

MA

samples exhibited antibacterial activities owing to the antibacterial potency of CS. Moreover, the CS grafted-acylated wool fabrics showed antifelting feature.

ED

The patchouli oil loaded CS–gelatin microcapsules were developed through the complex coacervation technique [185]. The morphology and surface were investigated by SEM micrographs displaying the microcapsules had regular spherical shapes in the range of

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1-20 μm. The thermal stability analysis exhibited that the microcapsules were stable below 190 ºC indicating the fabrics finish should be performed at 160 ºC. The loading capacity and encapsulation efficiency of the microcapsules were 30.31 and 50.69%, respectively. The

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microcapsules were grafted to cotton fabrics using the 2D resin dimethylol dihydroxyethylene urea as the crosslinking agent. The SEM images illustrated that the microcapsules were not only grafted onto the fabrics surfaces but also introduced in the fibers spaces. Furthermore, the creation of ether bonds among 2D resin and hydroxyl groups of cotton and/or hydroxyl moieties of the microcapsules was confirmed by FTIR spectra. The antibacterial capacities of the fabrics against Escherichia coli and Staphylococcus aureus were ~65% even after 25 times washing verifying their potential applications as antibacterial masks, bacteriostatic sheets and health-care clothes.

ACCEPTED MANUSCRIPT 5. Molecular dynamic (MD) simulations on pharmaceutical applications of chitosan Molecular dynamic simulation is a useful tool for researchers to investigate the structural and dynamical changes in atomic-scale resolution which are hardly observed in experimental efforts [186,187]. Moreover, the MD simulation is a powerful method and a valuable complement to experiment that can allow to avoid expensive experiments. Hence, MD simulations are commonly applied on drug delivery systems in order to examine the drug encapsulation at the molecular level [108].

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Follicle-stimulating hormone (FSH) is used in modern ovarian stimulation methods but it must be daily administered due to it has a short half-life. The cholesterol (ChS) modified

RI

CS nanogels are known as favorable controlled release protein delivery systems because they

SC

are able to decrease irreversible denaturation and aggregation of proteins [188]. The MD simulations were carried out for up to 200 ns on FSH encapsulation into ChS-CS nanogels

NU

and it was found that the main driving force for the creation of the ChS-CS nanogels was hydrophobic interactions occurred in water between the ChS–ChS fragments. Also, the ChSCS nanogel could form by the hydrogen bonds along with the hydrophobic interactions. Fig.

MA

49 exhibits the morphology and structure of the ChS-CS nanogel. The solvent-exposed hydrophobic patch in FSH is indicated in Fig. 50. The FSH encapsulation in the ChS-CS

ED

nanogels was a slow process which was motivated by the hydrophobic interactions happened between the FSH hydrophobic patch and the nanogel hydrophobic nanodomains. The total binding energy was decomposed per residue basis to recognize main residues that were

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contributed to the adsorption on ChS-CS. It was shown that the residues Phe17, Phe18, Val70, Met71, and Phe74 were the most important residues contributed to the FSH absorption to the ChS-CS nanogel by the non-polar solvation energy and the van der Waals interaction. All of

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these amino acids were positioned within the hydrophobic patch of FSHα confirming the most substantial contributions to the adsorption were from the hydrophobic patch region. Moreover, the total binding free energy for the FSH/CS-CTS nanogel was positive (+8.86). Considering the important role of drug delivery in the treatment of the central nervous system diseases, selecting an appropriate carrier is very crucial to have a higher drug efficiency. Thus, CS and poly(n-butylcyanoacrylate) (PBCA) were chosen as carriers in brain drug delivery because they have indicated good biodegradability, biocompatibility and low toxicity [189]. For this purpose, MD simulations were performed using these polymers with diverse polymerization degrees and Tacrine as the most familiar drug used for the treatment of Alzheimer's disease. Figs. 51 and 52 reveal the structures of various CS-Tacrine and PBCA-Tacrine systems, respectively which confirm the interaction between the Tacrine

ACCEPTED MANUSCRIPT molecule and PBCA chains becomes stronger when the polymer chain length is increased whereas it is decreased in the CS/Tacrine. Recently, sorption of L- and D-Tyrosine (Tyr) from aqueous solutions onto chiral CS membranes was evaluated by docking and MD calculations and a high adsorption with a noticeable

enantioselectivity

to

L-Tyr,

was

established

[190].

Furthermore,

different

enantiomers affinities were found for two adsorption regions within the polymeric matrix. It was indicated that Tyr adsorption decreased the membrane crystallinity and rearranged the

PT

chains. As well, the hydrated to anhydrous polymorph ratio was changed upon the adsorption due to water bound to CS was altered. The energy balance between the hydrogen bond

RI

formation, desolvation and the conformational changes caused an endothermic spontaneous

SC

process.

The stability and solubility of the CS film were measured by evaluating the interchain

NU

distances using a cut-off value of 0.35 nm. It was found that the sixteen CS chains remained close enough to each other so that they were assumed as a part of a single aggregate film during the entire simulation time. Fig. 53 indicates the simulation of CS-NaOH film in which

MA

three stages of the MD simulations from a crystal-like structure (0 ns) to a relax solvated film (50 ns) are depicted. Also, distances between the closest atoms in neighboring chains are computed horizontally and vertically. Although there were some fluctuations particularly

ED

between chains B and C (Fig. 3A–C), the total chain’s connectivity held the structure together. The distance between chains B and C showed the deviation from the average

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minimum distance of 0.2 nm occurred for most of chains. The temporary separations created holes/microcavities that allowed tyrosine and solvent molecules to move deeper in the film core. Therefore, swelling occurred and it was verified by increasing the number of water

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molecules near the central chains of the CS film in the first 5 ns of the simulation. Although the acetylation degree (20%) was low to have a soluble CS film, the modelled neutral environment lacked the electrostatic repulsion of protonated amino groups between CS polysaccharide chains, thus the polymer was remained aggregated as a solid membrane. The binding interactions of both tyrosine enantiomers (L-Tyr and D-Tyr) with CS film molecular docking calculations were carried out. Fig. 54 reveals the best energy ranked docking complexes for D-Tyr and L-Tyr. All tyrosine/CS-NaOH complexes exhibited the amino acid bound in a cleft between central film chains. Two possible binding regions were observed in this cleft including region 1 (placed deeper in the CS film core) and region 2 (located in the vicinity of the film surface) [190].

ACCEPTED MANUSCRIPT It is known that nanotechnology-based drug delivery systems are employed to increase the bioavailability and biological properties. Also, CS incorporated curcumin can be used as a biocompatible

alternative

for

metal

nanoparticles

to

prevent

biofilm

formation

of

Streptococcus mutans and plaque on teeth. The interactions between CS carrier and curcumin (a natural antibacterial agent) were examined by the simulation method [191]. The root mean square

deviation

(RMSD=26.81±0.1

Å)

and

the

root

mean

square

fluctuations

(RMSF=1.13±0.02 Å) for all of the complex atoms were relaxed after 4 ns. It was established

PT

that during the first interval (10 ns), a stable binding was occurred between the two sections. All bindings were disappeared from 10 to 20 ns but the curcumin was entrapped within the

RI

CS nanoparticles and no release was happened until 20 ns after which the curcumin started to

SC

release which confirmed the CS nanoparticle could carry the curcumin.

Chitosan oligosaccharide (COS) derivatives are attractive drug delivery systems

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because of their eminent low toxicity, outstanding biodegradability and biocompatibility. In an effort, salicylic acid-grafted chitosan oligosaccharide (COS/SA) was used to prepare paclitaxel (PTX) loaded nanoparticles [192]. Also, to realize the mechanism of the action of

MA

the PTX encapsulated COS/SA, MD simulations were conducted. The van der Waals and hydrophobic interactions were known as the major driving forces in the drug encapsulation.

ED

The hydrogen-bonding and electrostatic interactions have useful effects in the COS/SA aggregation. The solvent accessible surface area (SASA) and radial distribution function data showed that the COS/SA nanoparticles were extremely water soluble that could considerably

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increase the aqueous solubility of a hydrophobic drug. Different drug loading systems were examined and the best theoretical drug loading was obtained to be 10%w/w. A 20 ns run of three systems was performed under the same

AC C

conditions to investigate drug loading in which forty COS/SA chains were located near 5, 7, and 9 PTX molecules that were related to drug loadings of 7.3%, 10%, 12% (w/w), respectively. For the 12% drug loaded system, the 20 ns MD trajectory revealed that the 40 COS/SA chains did not spontaneously form a compact aggregated structure (Fig. 55). The average SASAs values for the nanoparticles in these three systems were 391.04, 391.59, and 432.41 nm2 , respectively. The SASAs at drug loadings of 7.3% and 10% nanoparticles were practically comparable but the SASA at the drug loading of 12% was significantly enhanced in comparison with the two other systems. Consequently, the best theoretical PTX drug loading for COS/SA drug carrier was ~10%. The ratio of PTX to COS/SA was experimentally fixed at 20% but the measured drug content was only 3.45%. The difference between the

ACCEPTED MANUSCRIPT simulation and the observation was related to the influence of the experimental operating conditions [192]. The influence of the CS nanoparticles was studied on the structure, dynamics and enzyme activity of trypsin [193]. It was found that the enzyme activity in complex with the nanoparticles was somewhat improved indicating the interactions between the enzyme and the nanoparticles but fluorescence spectroscopy did not demonstrate significant variations in the trypsin conformation in the presence of nanoparticles. The MD simulations were done to

PT

investigate the mechanism of interactions occurred between the nanoparticles and trypsin. The binding free energy data proved that the nonpolar interactions were the main forces for the

RI

creation of stable nanoparticle-trypsin complex.

SC

It was found that the total, hydrophilic and hydrophobic SASA values of the CS nanoparticles were rapidly decreased in the beginning the MD simulations and then reached

NU

the average value of 113.5, 15.7 and 97.8 nm2 , respectively (Fig. 56A). The decline in SASA of CS nanoparticles in the presence of water confirmed induced compactness of the molecules which caused restrictions in the interactions between the CS chains and water molecules.

MA

Also, the total number of hydrogen bonds formed between the CS and water molecules was diminished with time whereas the total number of the CS-CS hydrogen bonds was enhanced

ED

(Fig. 56B). in order to examine the influence of the CS nanoparticles on the water structure, the RDF between water oxygen atoms was obtained. Fig. 56C displays that the peak at 0.28 nm in the presence of the nanoparticles was sharper than that of the pure water. It was

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proposed that the CS nanoparticles increased the hydrogen bonds between the water molecules, hence strengthened the water structure and stabilized the nanoparticles [193]. Chitosan is a biocompatible and biodegradable carbohydrate biopolymer which is one

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of the most useful polymers in the pharmaceutical science. Moreover, various protein and peptide drugs are applied as therapeutic agents which may be exposed to temperature stresses in transporting and/or storage stages. Such stresses may cause the protein structure unstable, alter the active structure and destroy its therapeutic function thus limit their usage as fruitful drugs. To overcome such problems related to the protein drugs, diverse materials like natural or

synthetic

polymers

can

be

utilized

to

prepare

protein

loaded

biocompatible

nano/microspheres. Recently, MD simulations were performed to investigate the effect of CS with different deacetylation degrees on the stability of Interferon αII structure at high temperature and to compare the data with those of commonly used biocompatible synthetic polymers including polylactic-co-glycolic acid and polyethylene glycol [194]. Final structures of IFNαII-polymer complexes for all systems after 50 ns of simulation are displayed in Fig.

ACCEPTED MANUSCRIPT 57. The conformational variations occurred at high temperature (343 K) both in the presence and absence of polymers were compared to results obtained for the protein at normal temperature (300 K). It was indicated that low deacetylated CS and polylactic co-glycolic acid were more effective in protein stability at high temperature but polyethyleneglycol was penetrated into the protein and showed some instability of protein conformation.

6 Conclusion

PT

Polymeric pharmaceutical carriers (such as chitosan) are of great attention due to they have numerous advantages mainly in enhancing the effectiveness and safety of the

RI

pharmaceutics. Such systems can encapsulate both hydrophilic and hydrophobic active

SC

substances. Additionally, they can result in higher stability for the therapeutics against enzymatic and chemical degradation, greater drug impact in the target organ and more

genes

and

oligonucleotides

that

may

NU

bioavailability. They can hold active compounds such as drugs, vaccines, peptides, proteins, be adsorbed,

encapsulated,

covalently and/or

electrostatically bound to their surfaces or inside their matrices. Indeed, these materials

MA

especially polysaccharides have developed medical treatments. It is noteworthy that the CSbased systems are very appealing vehicles that are capable of releasing their active

ED

encapsulated compounds with the desired rate at targeted site in the body. Herein, the most important pharmaceutical applications of CS were discussed in numerous biomedical areas like

wound

healing,

tissue

engineering,

drug/gene

delivery,

protein

binding,

cell

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encapsulation, preparation of implants and contact lenses, bioimaging, food additives, food packaging and antibacterial textiles. Also, recent studies about the molecular dynamics simulation of chitosan in the pharmaceutical applications were reviewed. As most of the CS

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applications are yet at laboratory phase, additional investigations are necessary to utilize them as commercial pharmaceutics in clinical applications. Finally, it could be concluded that the CS-based materials are promising candidates for application in various pharmaceutical fields.

Acknowledgments Authors would like to appreciatively express their thanks to the research office of Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran for the financial support of this work.

Conflict of interests The authors do not have any personal or financial conflicts of interests.

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Scheme 1. A schematic indicating pharmaceutical applications of chitosan in various areas.

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Fig. 1. Het-Cam images for ocular irritation study treated with (A) 0.1N NaOH, (B) Normal saline (0.9% NaCl), (C) LFX-CS-NPopt in situ gel system.

(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 650–659).

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Fig. 2. Comparative histopathology images of treated groups (A) control, (B) LFX-CS-NPopt in situ gel.

(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 650–659).

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Fig. 3. Comparative confocal laser microscopy images of (A) LFX-CS-NPopt in situ gel, (B) LFX solution.

(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 650–659).

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Fig. 4. In vitro release of 5Fu from CS/Cc PECs (0.5 mg/mL solutions of CS and Cc,

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1:1(w/w) CS/Cc mass ratio, and 0 mM NaCl at pH 3.0) at pH 1.4 and pH 7.4. Each value is expressed by means ± SD (n=3).

(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 107 (2018) 397–405).

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Fig. 5. In vitro cellular uptake of CNPs in SCC7, U87, HT29, PC3, and A549 cells depending on various pH conditions. Confocal fluorescence microscopic images of each type of cancer cells after 30 min of treatment with CNPs (25 μg/mL) in the RPMI media (pH 6.0, 6.5, and 7.4). (Reprinted with permission from Elsevier, J. Control. Release 267 (2017) 223–231).

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Fig. 6. Distribution of CNPs in the mice transplanted SCC7, U87, HT29, PC3, A549 tumors. Time-dependent non-invasive in vivo NIRF imaging of the tumor-bearing mice after the injection of NIRF dye labeled CNPs (200 μg/μL head). (Reprinted with permission from Elsevier, J. Control. Release 267 (2017) 223–231).

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Fig. 7. Distribution of the intratumoral extracellular matrix in various tumors. Collagen matrix in tumor tissues stained blue with Masson's trichrome stain (bar = 150 μm). (B) Quantitative analysis of ECM contents in SCC7, U87, HT29, PC3, and A549 tumors; ECM contents in tumor tissue =blue stained collagen area / total area ×100 (%). (Reprinted with permission from Elsevier, J. Control. Release 267 (2017) 223–231).

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Fig. 8. HepG2 cells determined using Hoechst 33,258 stain. (a) cells untreated; (b) cells treated with free curcumin; (c) cells treated with curcumin loaded TCS/PEGDA injectable hydrogels without lysozyme (TP0); (d) cells treated with curcumin loaded TCS/PEGDA injectable hydrogels with lysozyme (TP3). (Reprinted with permission from Elsevier, Mater. Sci. Eng. C 83 (2018) 121–129).

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Fig. 9. In vivo antitumor effect of curcumin loaded TP3 on tumor-bearing nude mice. The optical pictures of tumor-bearing nude mice recorded at 0 day and 21 day of treatment (a), body weight changes (b) and tumor volume changes (c) from 0 day to 21 day. (Reprinted with permission from Elsevier, Mater. Sci. Eng. C 83 (2018) 121–129).

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Fig. 10. H&E staining of tumor, heart, liver, lung and kidney tissue sections from tumor-bearing nude mice (scale bar: 50 μm). (Reprinted with permission from Elsevier, Mater. Sci. Eng. C 83 (2018) 121–129).

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Fig. 11. (a) Relative cell viability of HepG2 cells treated with various concentrations of NPs (black), quercetin (red) and Q-NPs (blue). Trypan blue staining images of HepG2 cells without treatment (b), treated with 2 mg mL_1 of NPs (c), native quercetin (d), and Q-NPs (e). Data are shown as means ± SD of five separate experiments. Significant difference between treated and negative control groups is indicated at P<0.05 levels. (Reprinted with permission from Elsevier, LWT - Food Sci. Technol. 85 (2017) 37-44).

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Fig. 12. Gel retardation assays. (A) Gene retardation with the concentration of 10 µg/ml. (B) Gene retardation with the concentration of 15 µg/ml, and stability of them against heparin. miR-145 PECs of CS9 with CMD:CS ratios of 0.2 (1), 1 (2), 5 (3); CS18 with CMD:CS ratio of 0.2 (4), 1 (5), 5 (6); CS45 with CMD:CS ratio of 0.2 (7), 1 (8), 5 (9). The numbers 0, 0.1, 0.2, and 0.4 are the heparin concentrations (µg/ml). DL: DNA ladder 1 KD. C: naked plasmid. (Reprinted with permission from Elsevier, Carbohydr. Polym. 159 (2017) 66–75).

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Fig. 13. Green fluorescent protein (GFP) expression after transfection by miR-145 PECs. and DAPI staining of nucleus. (A) Confocal microscopy of non-transfected cells (a), and transfected cells by the nano PEC of CS45 with the CMD:CS 5 (b) and CS18 with the CMD:CS 1 (c); (B) Flowcytometery analysis. The miR-145 plasmid expresses both miR145and GFP. CS9: chitosan 9 KD, CS18: chitosan 18 KD, CS45: chitosan 45 KD, CMD:CS: the ratio of carboxymethyl dextran to chitosan. Data are presented as mean ± SD. (Reprinted with permission from Elsevier, Carbohydr. Polym. 159 (2017) 66–75).

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Fig. 14. Confocal microscopy of MCF7 treated with the Cy5 PECs composed of CS18

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with the CMD:CS ratios of 0.2 (A) and 1 (B). The merge image of nucleolus staining by DAPI and Cy5 PECs in the cells.

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Fig. 15. (a) Following 24 h incubation with polymers, cell viability was determined by the CellTiter-Glo®cell viability assay. Mean cell viability was normalized to non-treated controls, with the mean of n=3+SEM, from one representative experiments of three independent experiments. Statistical analysis was performed using two way ANOVA with tukey’s post hoc test, n.s.-not significant, *p<0.05, **p<0.01,***p<0.001. (b) Morphological characteristics of human fibroblast cells were visualized under the fluorescence microscope. Cells were also stained with the reagents in the LIVE/DEAD®Cell Viability/Cytotoxicity Assay Kit and visualized under the fluorescence microscope. Dead and live cells fluoresce red-orange and green, respectively. (Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).

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Fig. 15. Continued. (Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).

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Fig. 16. Evaluation of the specificity of targeted cell transfection by GnRH-CS/pDNA complexes. (a) Assessment of targeted gene transfer by GnRH-CS/pDNA compared to the unmodified CS/pDNA complexes carrying the GFP reporter gene using a range of polymer concentrations in the targeted cells expressing GnRH receptor and the non-targeted cells lacking GnRH receptor. (b) Transfection of 3D spheroids by polyplexes. The spheroids were transfected with polyplexes (i.e. GnRH-CS/pDNA and unmodified CS/pDNA).Representative images showing GFP expression in the monolayer cultures and spheroids were taken at day 3 post transfection. (Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).

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Fig. 16. Continued. (Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).

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Fig. 17. Mn-containing NPs were visualized and tracked by MRI. (A) Baseline T1-weighted image of coronal section through olfactory bulb and (B) baseline horizontal section showing the anatomical regions of interest. (C) T1-weighted MR of mouse 24 h after administration of mNPs showing enhanced Mn signal in coronal section of olfactory bulb, and (D) T1-weighted image signal in horizontal section including olfactory bulb, cerebral cortex, striatum and hippocampus. (E, F) Parcellation of brain regions (to demarcate brain structures) was performed to quantify Mn signal at 24 and 48 h. (G) Olfactory bulb; H) Cerebral Cortex; (I) Hippocampus; (J) Corpus Striatum. Mean Mn signal (±SEM, n = 3) was increased in all brain regions at 24 and 48 hrs compared to control mice (ie “no contrast”) after intranasal instillation. One-way ANOVA was performed for each brain region using Matlab Statistics Toolbox (Mathworks, Inc.). (Reprinted with permission from Elsevier, J. Drug Deliv. Sci. Technol. 43 (2018) 453–460).

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Fig. 17. Continued. (Reprinted with permission from Elsevier, J. Drug Deliv. Sci. Technol. 43 (2018) 453–460).

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Fig. 18. RFP expression in mouse brains following intranasal instillation of mNPs containing dsDNA encoding RFP. Mice were euthanized at 24 h for fluorescence histology and at 48 h for measurement of RFP DNA regional brain concentration. (A) Olfactory bulb. Left panel: Structure of the olfactory bulb is visualized with DAPI staining, which stains all cell nuclei. Middle panel: Tyrosine hydroxylase (TH) immunostaining in green identifies dopaminergic neurons located in the glomerulosa layer. Red identifies cells that express red fluorescent protein (RFP), indicating that the plasmid DNA encapsulated in the manganese-containing nanoparticles is expressed. Right panel: Merged image of DAPI, TH and RFP. onl=olfactory nerve layer; gl=glomerular layer; opl=outer plexiform layer; ml=mitral cell layer; gr=granular cell layer. (B) Corpus striatum. Left panel: neurons immunostained with antibodies against neuron specific nuclear protein (NeuN); Middle panel: RFP expression; right panel: merged image of NeuN and RFP. (C) Ventral striatum. Left panel: DAPI-stained nuclei and RFP expression; Right panel: magnified yellow box from panel on left shows RFP expression in cytoplasm distributed in a perinuclear pattern in many, but not all cells. (D) Regional brain concentrations of RFP DNA was determined at 48 hrs after nasal instillation Mn-nanoparticles containing DNA encoding RFP as described in methods section. Data are expressed as mean and SEM for each brain region (n = 4 mice). Y-axis indicates concentration of DNA (pg/mg tissue). Highest mean concentration of RFP DNA was observed in striatum with the lowest concentration found in olfactory bulb at this time point. (Reprinted with permission from Elsevier, J. Drug Deliv. Sci. Technol. 43 (2018) 453–460).

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Fig. 19. Confocal laser microscopy images of chondrocytes that were stained in the three gels (a)10:1 CS-HDA (b) 10:3 CS-HDA and (c) 10:5 CS-HDA with Calcein AM/ethidiumbromide (LIVE/DEAD assay) for ascertaining the viability of the cells that were encapsulated. Live cells fluoresce green and red cells fluoresce red. Scale bar = 10 µm. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).

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Fig. 20. (a) Fluorescence images showing Type II Collagen staining of chondrocytes encapsulated in 10:1 CS-HDA, 10:3 CS-HDA and 10:5 CS-HDA gels at 7 day, 14 day and 28 day. Cells were stained for type II collagen green and nuclei (blue). Scale bar for 7 day = 10 µm, 14 day = 5 µm and 28 day = 2 µm. (b) Fluorescence images showing Type I Collagen staining of chondrocytes encapsulated in 10:1 CS-HDA, 10:3 CS-HDA and 10:5 CS-HDA gels at 7 day (a, b and c),14 day (d–f) and 28 day (g–i). Cells were stained for Type I collagen (red) and nuclei (blue). Scale bar for (a–c) = 10 µm and for (d–i) = 20 µm. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).

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Fig. 20. Continued. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).

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Fig. 21. (a) Sirius red staining for collagen performed on the chondrocyte encapsulated gels at 7 day (a, b and c),14 day (d–f) and 28 day (g–i). Scale bar = 50 µm. (b) Alcian blue staining for collagen performed on the chondrocyte encapsulated gels at 7 day (a, b and c),14 day (d, e, f) and 28 day (g, h, i). Scale bar = 50 µm. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).

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Fig. 21. Continued.

(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).

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Fig. 22. Release of ASCs from the border of the hydrogels placed on a layer of Hs68 fibroblast and in vitro wound healing assay. (A) Scheme of the experimental design. (B) Double immunofluorescence of ASCs (green) and fibroblasts (red), the dash line represents the border of chitosan or chitosan/gelatin hydrogels. The number of ASC migrated beyond the dash line was estimated at 24 and 48 h after applying the hydrogel (image analysis performed on 4 random fields per sample; n=3 samples/condition). (C) After scratching confluent fibroblasts with a pipette tip, the cell-free zone was photographed at 24 and 48 h. Area in the cell-free zone covered by the migrated cells was measured (scale bars: 100 µm; image analysis performed on 4 random fields per sample; n=3 samples/condition; *p<0.05, compared to the group treated with chitosan hydrogel; # p<0.05 between the indicated two groups). (Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).

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Fig. 22. Continued. (Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).

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Fig. 23. In vitro tube formation assay. (A) Images of endothelial cells after 4 h incubation with hydrogels or ASC-encapsulated hydrogels at the periphery of each well (scale bars: 100 µm). (B) Branching points per power field were compared among different groups (image analysis performed on 4 random fields per sample; n=3 samples/condition; *p<0.05, compared to the group treated with chitosan or chitosan/gelatin hydrogel). (Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).

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Fig. 24. Chick embryo chorioallantoic membrane (CAM) assay. (A) Photographs of CAMs after treatment with hydrogels or ASC-encapsulated hydrogels, which were loaded on the CAMs of day 9 chick embryos. After 72 h of incubation, CAMs were excised and photographed (scale bars: 100 lm). (B) Blood vessel formation on CAM was quantified by measuring the area covered by capillaries and counting the number of blood vessel branch points (image analysis performed on 10 random fields per sample; n=3 samples/condition; *p<0.05, compared to any of the other groups). (Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).

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Fig. 24. Continued. (Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).

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Fig. 25. Delivery of ASC-encapsulated chitosan/gelatin hydrogel enhanced angiogenesis in a mice cutaneous wound model. (A) Double immunofluorescent staining of HNA and an endothelial marker CD31 in the day 5 wound sections. White arrows indicate co-localization of the HNA and CD31 immunofluorescence, indicating human ASCs incorporated into vasculature structure (scale bar: 100 µm). (B) The number of HNA + cells per power field was significantly higher in wound sections that received ASC encapsulated chitosan/gelati n hydrogel. (C) The ratio of CD31 + area was also significantly larger in the group of ASCencapsulated chitosan/gelatin hydrogel (image analysis performed on 10 random fields per sample; n=4 samples/condition; #p<0.05, between the indicated two groups). (Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).

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Fig. 26. Chemical structures of chitosan and BSA and HSA with tryptophan residues in green color. (Reprinted with permission from Elsevier, Colloid. Surf. B Biointerfaces 125 (2015) 309–317).

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Fig. 27. Fluorescence emission spectra of BSA or HSA (10 µM) in acetate buffer pH 5–6 at 298.15 K, in the presence of chitosan nanoparticles with the following concentration:0 (a), 25 (b), 50 (c), 80 (d) and 120 µM (e) for chitosan-15; 0 (a), 5 (b), 10 (c), 15 (d), 20 (e) and 30 µM (f) for chitosan-100 and chitosan-200. (Reprinted with permission from Elsevier, Colloid. Surf. B Biointerfaces 125 (2015) 309–317).

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Fig. 28. (a) Typical immunofluorescent staining images of α-actinin, and (b) Tn-I synthesized by cardiomyocytes after incubation for 15 days on composite PLA/chitosan electrospun nanofibers. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 1130–1137).

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Fig. 29. Scanning electron micrographs of 3T3 on (A) 4G2C, (B) 4G2C10HY, (C) 4G2C20HY, (D) 4G2C30HY and (E) 4G2C50HY cryogels matrix after 10 days of culture at 5000x magnification. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 366–378).

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Fig. 30. Scanning electron micrographs of SaOS-2 on (A) 4G2C, (B) 4G2C10HY, (C) 4G2C20HY, (D) 4G2C30HY and (E) 4G2C50HY cryogels matrix after 10 days of culture at 5000x magnification. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 366–378).

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Fig. 31. (i) Steps of surgical procedures: (a) Exposed rat calvarium (b) Selected area on

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parietal bone (c) Round critical size defect (8 mm). Defect filled with (d) CHA-1RS nanocomposite scaffold (e) Cerabone (f) sham control. (g) Periostium sutured with vicryl (h) Skin stitched with silk. (ii) Histopathology of bone: Overviews and magnified images of the

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black-lined squares of the sections at 4 weeks post-implantation (a-c): (a) Sham control group (b) Cerabone Group, (c) CHA-1RS group. Stains H & E and SR; initial magnification x200. [OB: Osteoblasts, CF: Collagen Fibre, CB: Cerabone, IB: Immature bone, MB: Mature Bone, NC: Nanocomposite scaffold, FT: Fibrous tissue]. (Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).

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Fig. 31. Continued. (Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).

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Fig. 32. Histopathology of bone: Overviews and magnified images of the black-lined squares of the sections at 8 weeks post-implantation (a-c): (a) Sham control group (b) Cerabone group (c) CHA-1RS group. Stain H & E and SR. Initial magnification x200. Histopathological results of Kidney (d,e) and liver (f,g). Left panel (d & f) are from control group and right panel (e & g) from CHA-1RS group. H & E stain, initial magnification x200. [OC: Osteoclasts, OB: Osteoblasts, CF: Collagen Fibre, CB: Cerabone, MB: Mature Bone, FT: Fibrous tissue]. (Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).

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Fig. 33. (i) Radiographs: X ray and RVG 4 weeks: (a,g) sham control, (b,h) cerabone, (c,i) CHA-1RS group, and 8 weeks post-implantation: (d,j) sham control, (e,k) cerabone, (f,l) CHA-1RS respectively. Harvested calvaria viewed from inside: (m) Sham control, (n) Cerabone, (o) CHA-1RS after 8 weeks post-implantation with arrows indicating defect site. (ii) Quantitative analysis of the Gain in Bone density (GBD)% of CHA-1RS, CB and Sham control groups after the time period of 4 and 8 weeks. (Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).

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Fig. 33. Continued. (Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).

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Fig. 34. Histopathology of bone from CHA-1RS group after 2 weeks post implantation: Overview and magnified images of the black-lined squares (a) Stain H & E and SR, initial magnification x200. (b) Bone defect site in situ (Top view) and (c) the bone defect site as seen from inside (Bottom view) and (d) its X-Ray and (e) RVG radiographs with arrows indicating defect site. [OB: Osteoblasts, NC: Nanocomposite scaffold, HB: Host Bone, WB: Woven Bone, CT: Connective Tissue, DM: Defect Margin]. (Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).

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Fig. 35. (a) Biofilm formation by S. mutans in the presence of chitosan and conjugate Ag nanoparticle. (b) ImageJ analysis of biofilm biomass. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 790-797).

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Fig. 36. Phase contrast Image of HGF before and after treatment with nanoparticle materials. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 790-797).

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Fig. 37. Cell viability studies using XTT assay. (Reprinted with permission from Elsevier, Eur. J. Pharm. Biopharm. 109 (2016) 61–71).

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Fig. 38. Slit-lamp photograph of (a) eye after wearing functional contact lens for 35 days; (b) untreated eye after 35 days. Light microscope observation (HE stain) of (c) eye after wearing functional contact lens for 35 days; and (d) untreated eye after 35 days eye at day 35 after surgery. (Reprinted with permission from Elsevier, J. Mech. Behav. Biomed. Mater. 64 (2016) 43–52).

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Fig. 39. In vivo biocompatibility studies of C7P3 by subcutaneous implantation in rat model (a) subcutaneous pouch (b) implantation of C7P3 scaffold (c) retrieval of tissue integrated with C7P3 in back of albino Wister rat; (d) H&E (Hematoxylin & Eosin); (e)MT (Masson Trichrome) and (f) TB (Toluidine Blue) staining of subcutaneous tissue harvested from the implanted site; Black line demarcates between host and scaffold, black arrows represent blood vessel and star represents mast cells. (Reprinted with permission from Elsevier, Mater. Sci. Eng. C 81 (2017) 133–143).

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Fig. 40. Photographs (a) and closure rate (b) of wounds treated with Tegaderm™ (control) and C7P3 during the wound healing process for 21 days. Arrow represents implanted scaffold, Y-error bars represent standard deviation. (Reprinted with permission from Elsevier, Mater. Sci. Eng. C 81 (2017) 133–143).

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Fig. 41. The healing promoting effect of CCA composite dressing on seawater immersion wound of rat. A: The wound healing image of the dressings. B: The wound healingratio statistics of the dressings. compared to gauze group *P<0.05), ** P<0.01. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 107 (2018) 93–104).

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Fig. 42. The effects of different dressings on the healing process of rat dorsalis skin wounds on the fifth, eighth, 11th , and 13th days post-surgery examined by H&E staining (×200). (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 107 (2018) 93–104).

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Fig. 43. (a) Average volume of mice-bearing HeLa tumors at different time points after intravenous injection of PBS, free DOX, Ag2 S@CS and Ag2 S(DOX)@CS nanospheres via tail vein. Statistical significance: *P<0.05. (b) Body weight changes of the tumor-born mice after treatment with PBS, free DOX, Ag2 S@CS and Ag2 S(DOX)@CS nanospheres. The data are presented as average±standard deviation (n=3). (c) ICP-MS analysis of tumor and five major organs of the mice sacrificed at different time points after injection of Ag2 S(DOX)@CS. The data are presented as average±standard deviation (n=5). Statistical significance: *P<0.05. (d) In vivo NIR images of a nude mouse at 6 h (i), 12 h (ii)and 24 h (iii) after injection of the Ag2 S(DOX)@CS nanospheres; ex vivo NIR image of the tumor (iv) and the organs (v) harvested from the sacrificed nude mouse. (Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 325–334).

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Fig. 43. Continued. (Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 325–334).

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Fig. 44. Inhibition of Staphylococcus aureus and Escherichia coli following antibiotic disk diffusion assay. GEL:gelatin, CS:chitosan, R:Rosemary,C:cinnamon, B:boldo and G:guaraná. A:GEL50:CS50 films with extracts added against S. aureus B: GEL50:CS50 films with extracts added against E. coli. (Reprinted with permission from Elsevier, Food Biosci. 16 (2016) 17–25).

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Fig. 45. Image of flexible antimicrobial C-2 pouch. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 97 (2017) 382–391).

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Fig. 46. Image of packed meat samples in C-2 pouches (A1, A2 & A3) and plastic bags (B1, B2 & B3). (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 97 (2017) 382–391).

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Fig. 47. HR-SEM images of (a) CS-NS composite (b) Untreated cotton fabric (c) CS-NS composite coated cotton (d) CS-NS composite coated cotton fabric with glutaraldehyde and (e) CS-NS composite coated cotton fabric with citric acid. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1890–1896).

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Fig. 48. SEM at low and high magnification of (A) untreated cotton fabrics, cotton fabric treated with (B) CS solution, (C) CS/AgNPs nanocomposite and (D) CS/AgNPs/clay nanocomposite. (Reprinted with permission from Elsevier, Carbohydr. Polym. 182 (2018) 29–41).

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Fig. 49. The morphology and structure of the ChS-CS nanogel. It is observed that there is more than one hydrophobic nanodomain in the nanogel structure. Additionally, the complexation process was mainly occurred by the hydrophobic interactions between the FSH hydrophobic patch and the cholesterol groups in the hydrophobic nanodomains existing in the nanogel. (Reprinted with permission from Elsevier, Eur. J. Pharm. Sci. 107 (2017) 126-137).

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Fig. 50. The solvent-exposed hydrophobic patch in FSH. (Reprinted with permission from Elsevier, Eur. J. Pharm. Sci. 107 (2017) 126-137).

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Fig. 51. The structures of various CS-Tacrine systems in which the polymer chains and Tacrine molecules are indicated in green and pink colors, respectively.

(Reprinted with permission from Elsevier, Eur. J. Pharm. Sci. 82 (2016) 79–85).

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Fig. 52. The structures of different PBCA-Tacrine systems in which the polymer chains and Tacrine molecules are indicated in blue and pink colors, respectively. (Reprinted with

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permission from Elsevier, Eur. J. Pharm. Sci. 82 (2016) 79–85).

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Fig. 53. Simulation of CS-NaOH film. [A] Three stages of the MD simulation from a crystallike structure (0 ns) to a relax solvated film (50 ns). Polysaccharide chains are labelled with letters from A to P. Distances between the closest atoms of neighbor chains computed horizontally [B] or vertically [C]. (Reprinted with permission from Elsevier, Carbohydr. Polym. 206 (2019) 57–64).

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Fig. 53. Continued. (Reprinted with permission from Elsevier, Carbohydr. Polym. 206 (2019) 57–64).

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Fig. 54. Best energy ranked docking complexes for D-Tyr (purple sticks) [A] and L-Tyr (turquoise sticks) [B]. Dashed yellow lines represent hydrogen bonds. (Reprinted with permission from Elsevier, Carbohydr. Polym. 206 (2019) 57–64).

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Fig. 55. The last snapshot of the system after 20 ns indicating drug loading values of (a) 7.3%, (b) 10% and (c) 12%. (Reprinted with permission from Elsevier, Biomaterials 34 (2013) 1843-1851).

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Figure 56. (A) The total, hydrophobic and hydrophilic SASA values of the chitosan nanoparticles during the MD simulation. (B) The total number of hydrogen bonds between the chitosan molecules and the water molecules to compare with the total number of the chitosanchitosan hydrogen bonds. (C) Radial distribution function (RDF) between water oxygen atoms to study the effect of the chitosan nanoparticles on the water structure. (Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 902-909).

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Fig. 57. Final 3D structure of protein-polymer complexes; (A) before simulation and (B) after simulation. (Reprinted with permission from Elsevier, Carbohydr. Polym. 203 (2019) 52-59).

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Numerous pharmaceutical applications of chitosan-based compounds were reviewed.

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Chitosan was applied in pharmaceutics/drug/gene delivery and cell encapsulation.

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Chitosan was used in wound healing, tissue engineering, bioimaging and food industry. Chitosan was applied in binding to protein drugs, contact lenses and implants.

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Molecular dynamics simulations were done on pharmaceutical applications of

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